Fermentative carotenoid production

ABSTRACT

Novel proteins of microorganism E-396 (FERM BP-4283) and the DNA sequences which encode these proteins have been discovered to provide an improved biosynthetic pathway from farnesyl pyrophosphate and isopentyl pyrophosphate to various carotenoids, especially zeaxanthin, astaxanthin, adonixanthin and canthaxanthin.

BACKGROUND OF THE INVENTION

[0001] Over 600 different carotenoids have been described fromcarotenogenic organisms found among bacteria, yeast, fungi and plants.Currently only two of them, β-carotene and astaxanthin are commerciallyproduced in microorganisms and used in the food and feed industry.β-carotene is obtained from algae and astaxanthin is produced in Pfaffiastrains which have been generated by classical mutation. However,fermentation in Pfaffia has the disadvantage of long fermentation cyclesand recovery from algae is cumbersome. Therefore it is desirable todevelop production systems which have better industrial applicability,e.g. can be manipulated for increased titers and/or reduced fermentationtimes.

[0002] Two such systems using the biosynthetic genes form Erwiniaherbicola and Erwinia uredovora have already been described in WO91/13078 and EP 393 690, respectively. Furthermore, three β-caroteneketolase genes (β-carotene β4-oxygenase) of the marine bacteriaAgrobacterium aurantiacum and Alcaligenes strain PC-1 (crtW) [Misawa,1995, Biochem. Biophys. Res. Com. 209 867-876][Misawa, 1995, J.Bacteriology 177, 6575-6584 and from the green algae Haematococcuspluvialis (bkt) [Lotan, 1995, FEBS Letters 364, 125-128][Kajiwara, 1995,Plant Mol. Biol. 29, 343-352] have been cloned. E. coli carrying eitherthe carotenogenic genes (crtE, crtB, crtY and crtI) of E. herbicola[Hundle, 1994, MGG 245, 406-416] or of E. uredovora and complementedwith the crtW gene of A. aurantiacum [Misawa, 1995] or the bkt gene ofH. pluvialis [Lotan, 1995][Kajiwara, 1995] resulted in the accumulationof canthaxanthin (β,β-carotene4,4′-dione), originating from theconversion of β-carotene, via the intermediate echinenone(β,β-carotene4-one).

[0003] Introduction of the above mentioned genes (crtW or bkt) into E.coli cells harbouring besides the carotenoid biosynthesis genesmentioned above also the crtZ gene of E. uredovora [Kajiwara,1995][Misawa, 1995], resulted in both cases in the accumulation ofastaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione). The resultsobtained with the bkt gene, are in contrast to the observation made byothers [Lotan, 1995], who using the same experimental set-up, butintroducing the H. pluvialis bkt gene in a zeaxanthin(β,β-carotene-3,3′-diol) synthesising E. coli host harbouring thecarotenoid biosynthesis genes of E. herbicola, a close relative of theabove mentioned E. uredovora strain, did not observe astaxanthinproduction.

SUMMARY OF THE INVENTION

[0004] Novel proteins of microorganism E-396 (FERM BP-4283) and the DNAsequences which encode these proteins have been discovered which providean improved biosynthetic pathway from farnesyl pyrophosphate andisopentyl pyrophosphate to various carotenoids, especially zeaxanthin,astaxanthin, adonixanthin and canthaxanthin.

BRIEF DESCRIPTION OF THE FIGURES

[0005]FIG. 1: The biosynthesis pathway for the formation or carotenoidsof Flavobacterium sp. R1534 is illustrated explaining the enzymaticactivities which are encoded by DNA sequences of the present invention.

[0006]FIG. 2: Southern blot of genomic Flavobacterium sp. R1534 DNAdigested with the restriction enzymes shown on top of each lane andhybridized with Probe 46F. The arrow indicated the isolated 2.4 kb XhoI/PstI fragment.

[0007]FIG. 3: Southern blot of genomic Flavobacterium sp. R1534 DNAdigested with ClaI or double digested with ClaI and HindIII. Blots shownin Panel A and B were hybridized to probe A or probe B, respectively(see examples). Both ClaI/HindIII fragments of 1.8 kb and 9.2 kb areindicated.

[0008]FIG. 4: Southern blot of genomic Flavobacterium sp. R1534 DNAdigested with the restriction enzymes shown on top of each lane andhybridized to probe C. The isolated 2.8 kb SalI/HindIII fragment isshown by the arrow.

[0009]FIG. 5: Southern blot of genomic Flavobacterium sp. R1534 DNAdigested with the restriction enzymes shown on top of each lane andhybridized to probe D. The isolated BclI/SphI fragment of approx. 3 kbis shown by the arrow.

[0010]FIG. 6: Physical map of the organization of the carotenoidbiosynthesis cluster in Flavobacterium sp. R1534, deduced from thegenomic clones obtained. The location of the probes used for thescreening are shown as bars on the respective clones.

[0011]FIG. 7: Nucleotide sequence of the Flavobacterium sp. R1534carotenoid biosynthesis cluster and its flanking regions (SEQ ID NO: 1).The nucleotide sequence is numbered from the first nucleotide shown (seeBamHI site of FIG. 6). The deduced amino acid sequence of the ORF's(orf-5, orf-1, crtE, crtB, crtI, crtY, crtZ and orf-16) are shown withthe single-letter amino acid code. Arrow (→) indicate the direction ofthe transcription; asterisks, stop codons.

[0012]FIG. 8: Protein sequence of the GGPP synthase (crtE) ofFlavobacterium sp. R1534 (SEQ ID NO: 2) with a MW of 31331 Da.

[0013]FIG. 9: Protein sequence of the prephytoene synthetase (crtB) ofFlavobacterium sp. R1534 (SEQ ID NO: 3) with a MW of 32615 Da.

[0014]FIG. 10: Protein sequence of the phytoene desaturase (crtI) ofFlavobacterium sp. R1534 (SEQ ID NO: 4) with a MW of 54411 Da.

[0015]FIG. 11: Protein sequence of the lycopene cyclase (crtY) ofFlavobacterium sp. R1534 (SEQ ID NO: 5) with a MW of 42368 Da.

[0016]FIG. 12: Protein sequence of the β-carotene hydroxylase (crtZ) ofFlavobacterium sp. R1534 (SEQ ID NO: 6) with a MW of 19282 Da.

[0017]FIG. 13: Recombinant plasmids containing deletions of theFlavobacterium sp. R1534 carotenoid biosynthesis gene cluster.

[0018]FIG. 14: Primers used for PCR reactions (SEQ ID NOs: 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, and 18). The underlined sequence is therecognition site of the indicated restriction enzyme. Small capsindicate nucleotides introduced by mutagenesis. Boxes show theartificial RBS which is recognized in B. subtilis. Small caps in boldshow the location of the original adenine creating the translation startsite (ATG) of the following gene (see original operon). All the ATG's ofthe original Flavobacter carotenoid biosynthetic genes had to bedestroyed to not interfere with the rebuild transcription start site.Arrows indicate start and ends of the indicated Flavobacterium R1534 WTcarotenoid genes.

[0019]FIG. 15: Linkers used for the different constructions (SEQ ID NOs:19, 20, 21, 22, 23, 24, 25, and 26). The underlined sequence is therecognition site of the indicated restriction enzyme. Small capsindicate nucleotides introduced by synthetic primers. Boxes show theartificial RBS which is recognized in B. subtilis. Arrow indicate startand ends of the indicated Flavobacterium carotenoid genes.

[0020]FIG. 16: Construction of plasmids pBIIKS(+)-clone59-2, pLyco andpZea4.

[0021]FIG. 17: Construction of plasmid p602CAR.

[0022]FIG. 18: Construction of plasmids pBIIKS(+)-CARVEG-E and p602CARVEG-E.

[0023]FIG. 19: Construction of plasmids pHF13-2CARZYIB-EINV andpHP13-2PN25ZYIB-EINV.

[0024]FIG. 20: Construction of plasmid pXI12-ZYIB-EINVMUTRBS2C.

[0025]FIG. 21: Northern blot analysis of B. subtilis strainBS1012::ZYIB-EINV4. Panel A: Schematic representation of a reciprocalintegration of plasmid, pXI12-ZYIB-EINV4 into the levan-sucrase gene ofB-subtilis. Panel B: Northern blot obtained with probe A PCR fragmentwhich was obtained with CAR 51 and CAR 76 and hybridizes to the 3′ endof crtZ and the 5′ end or crtY). Panel C: Northern blot obtained withprobe B (BamHI-Xhol fragment isolated from plasmid pBIIKS(+)-crtE/2 andhybridizing to the 5′ part of the crtE gene).

[0026]FIG. 22: Schematic representation of the integration sites ofthree transformed Bacillus subtilis strains: BS1012::SFCO,BS1012::SFCOCAT1 and BA1012::SFCONEO1. Amplification of the syntheticFlavobacterium carotenoid operon (SFCO) can only be obtained in thosestrains having amplifiable structures. Probe A was used to determine thecopy number of the integrated SFCO. Erythromycine resistance gene(ermAM), chloramphenicol resistance gene (cat), neomycine resistancegene (neo), terminator of the cryT gene of B. subtilis (cryT),levan-sucrase gene (sac-B 5′ and sac-B 3′), plasmid sequences of pXI12(pXI12), promoter originating from site I of the veg promoter complex(PvegI).

[0027]FIG. 23: Construction of plasmids pXI12-ZYIB-EINV4MUTRBS2CNEO andpXI12-ZYIB-EINV4MUTRBS2CCAT.

[0028]FIG. 24: Complete nucleotide sequence of plasmid pZea4 (SEQ ID NO:27).

[0029]FIG. 25: Synthetic crtW gene of Alcaligenes PC-1 (SEQ ID NO: 28).The translated protein sequence (SEQ ID NO: 29) is shown above thedouble stranded DNA sequence. The twelve oligonucleotides (crtW1-crtW12)used for the PCR synthesis are underlined.

[0030]FIG. 26: Construction of plasmid pBIIKS-crtEBIYZW. TheHindIII-Pm1I fragment of pALTER-Ex2-crtW, carrying the synthetic crtWgene, was cloned into the HindIII and MluI (blunt) sites. PvegI and Ptacare the promoters used for the transcription of the two opera. The CoIE1replication origin of this plasmid is compatible with the p15A originpresent in the pALTER-Ex2 constructs.

[0031]FIG. 27: Relevant inserts of all plasmids constructed in Example7. Disrupted genes are shown by //. Restriction sites: S=SacI, X=XbaI,H=HindIII, N=NsiI, Hp=HpaI, Nd=NdeI.

[0032]FIG. 28: Reaction products (carotenoids) obtained from β-caroteneby the process of the present invention.

[0033]FIG. 29: Isolation of the crt cluster of the strain E-396. GenomicDNA of E-396 was digested overnight with different combinations ofrestrictions enzymes and separated by agarose gel electrophoresis beforetransferring the resulting fragments by Southern blotting onto anitrocellulose membrane. The blot was hybridised with a ³²P labelled 334bp fragment obtained by digesting the aforementioned PCR fragmentJAPclone8 with BssHII and MluI. An approx. 9,4 kb EcoRI/BamHI fragmenthybridizing to the probe was identified as the most appropiate forcloning since it is long enough to potentially carry the complete crtcluster. The fragment was isolated and cloned into the EcoRI and BamHIsites of pBluescriptIIKS resulting in plasmid pJAPCL544.

[0034]FIG. 30 Shows the sequence obtained containing the crtW_(E396)(from nucleotide 40 to 768) and crtZ_(E396) (from nucleotide 765 to1253) genes of the bacterium E-396 (SEQ ID NO: 30).

[0035]FIG. 31: The nucleotide sequence of the crtW_(E396) gene (SEQ IDNO: 31).

[0036]FIG. 32: The amino acid sequence encoded by the crtW_(E396) (SEQID NO: 32) gene shown in FIG. 31.

[0037]FIG. 33: The nucleotide sequence of the crtZ_(E396) (SEQ ID NO:33) gene.

[0038]FIG. 34: The amino acid sequence (SEQ ID NO: 34) encoded by thecrtZ_(E396) gene shown in FIG. 33.

[0039]FIG. 35: Diagram of plasmid pUC18-E396crtWZPCR.

[0040]FIG. 36: Construction of plasmid pBIIKS-crtEBIY[E396WZ].

[0041]FIG. 37: Construction of plasmid pBIIKS-crtEBIY[E396W]DZ which hasa truncated non-functional crtZ gene.

[0042]FIG. 38: 463 bp PstI-BamHI fragment (SEQ ID NO: 35) originatingfrom the 3′ end of the insert of pJAPCL544 (FIG. 29) highlighted a ˜1300bp-long PstI-PstI fragment. This fragment was isolated and cloned intothe PstI site of pBSIIKS(+) resulting in plasmid pBSIIKS-#1296. Thesequence of the insert is shown (small cap letters refer to new sequenceobtained. Capital letters show the sequence also present in the 3′ ofthe insert of plasmid pJAPCL544).

[0043]FIG. 39: The DNA sequence of the complete crtE_(E396) gene (SEQ IDNO: 36).

[0044]FIG. 40: The amino acid sequence encoded by the crtE_(E396) gene(SEQ ID NO: 37) shown in FIG. 39 (SEQ ID NO: 36).

[0045]FIG. 41: Construction of plasmid carrying the complete crt clusterof E-396 (pE396CARcrtW-E).

[0046]FIG. 42: Construction of plasmid pRSF1010-Amp^(r).

[0047]FIG. 43: Construction of plasmids RSF1010-Amp^(r)-crt1 andRSF1010-Ampr-crt2.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Novel proteins of microorganism E-396 (FERM BP-4283) and the DNAsequences which encode these proteins have been discovered which providean improved biosynthetic pathway from farnesyl pyrophosphate andisopentyl pyrophosphate to various carotenoids, especially zeaxanthin,astaxanthin, adonixanthin and canthaxanthin.

[0049] One aspect of the invention is a polynucleotide comprising a DNAsequence which encodes the GGPP synthase (crtE_(E396)) (SEQ ID NO: 37)of microorganism E-396, said polynucleotide being substantially free ofother polynucleotides of microorganism E-396. Also encompassed by thisaspect of the present invention is a polynucleotide comprising a DNAsequence which is substantially homologous to said DNA sequence. SaidGGPP synthase catalyzes the condensation of farnesyl pyrophosphate andisopentyl pyrophosphate to obtain geranylgeranyl pyrophosphate, acarotenoid precursor. The preferred GGPP synthase has the amino acidsequence of FIG. 40 (SEQ ID NO: 37), and the preferred DNA sequenceencodes said amino acid sequence. The especially preferred DNA sequenceis shown in FIG. 39 (SEQ ID NO: 36).

[0050] This aspect of the present invention also includes a vectorcomprising the aforesaid polynucleotide, preferably in the form of anexpression vector. Furthermore this aspect of the present invention alsoincludes a recombinant cell comprising a host cell which is transformedby the aforesaid polynucleotide or vector which contains such apolynucleotide. Preferably said host cell is a prokaryotic cell and morepreferably said host cell is E. coli or a Bacillus strain. However, saidhost cell may also be a eukaryotic cell, preferably a yeast cell or afungal cell.

[0051] Finally this aspect of the present invention also comprises aprocess for the preparation of geranylgeranyl pyrophosphate by culturingsaid recombinant cell of the invention containing farnesyl pyrophosphateand isopentyl pyrophosphate in a culture medium under suitable cultureconditions whereby said GGPP synthase is expressed by said cell andcatalyzes the condensation of farnesyl pyrophosphate and isopentylpyrophosphate to geranylgeranyl pyrophosphate, and isolating thegeranylgeranyl pyrophosphate from such cells or the culture medium.

[0052] Another aspect of the present invention is a polynucleotidecomprising a DNA sequence which encodes said β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 34), said polynucleotidebeing substantially free of other polynucleotides of microorganismE-396. Also encompassed by this aspect of the present invention is apolynucleotide comprising a DNA sequence which is substantiallyhomologous to said DNA sequence. Said β-carotene hydroxylase catalyzesthe hydroxylation of β-carotene to produce the xanthophyll, zeaxanthin.The preferred β-carotene hydroxylase has the amino acid sequence of FIG.34 (SEQ ID NO: 34), and the preferred DNA sequence is one which encodessaid amino acid sequence. The especially preferred DNA sequence is a DNAsequence comprising the sequence shown in FIG. 33 (SEQ ID NO: 33).

[0053] This aspect of the present invention also includes a vectorcomprising the aforesaid polynucleotide, preferably in the form of anexpression vector. Furthermore this aspect of the present invention alsoincludes a recombinant cell comprising a host cell which is transformedby the aforesaid polynucleotide or vector which contains such a DNAsequence. Preferably said host cell is a prokaryotic cell and morepreferably said host cell is E. coli or a Bacillus strain. However, saidhost cell may also be a eukaryotic cell, preferably a yeast cell or afungal cell.

[0054] Finally this aspect of the present invention also comprises aprocess for the preparation of zeaxanthin by culturing said recombinantcell of the invention containing β-carotene in a culture medium undersuitable culture conditions whereby said β-carotene hydroxylase isexpressed by said cell and catalyzes the hydroxylation of β-carotene toproduce the xanthophyll, zeaxanthin, and isolating the zeaxanthin fromsuch cells or the culture medium.

[0055] Another aspect of the present invention is a polynucleotidecomprising a DNA sequence which encodes said β-carotene hydroxylase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32), said polynucleotidebeing substantially free of other polynucleotides of microorganismE-396. Also encompassed by this aspect of the present invention is apolynucleotide comprising a DNA sequence which is substantiallyhomologous to said DNA sequence. Said β-carotene β4-oxygenase catalyzesthe hydroxylation of β-carotene to produce the echinenone, and, with thefurther catalysis of echinenone by the enzyme encoded by crtW_(E396), tocanthaxanthin. The preferred β-carotene β4-oxygenase has the amino acidsequence of FIG. 32 (SEQ ID NO: 32), and the preferred DNA sequence isone which encodes said amino acid sequence. The especially preferred DNAsequence is a DNA sequence comprising the sequence shown in FIG. 31 (SEQID NO: 31).

[0056] This aspect of the present invention also includes a vectorcomprising the aforesaid polynucleotide, preferably in the form of anexpression vector. Furthermore this aspect of the present invention alsoincludes a recombinant cell comprising a host cell which is transformedby the aforesaid polynucleotide or vector which contains such a DNAsequence. Preferably said host cell is a prokaryotic cell and morepreferably said host cell is E. coli or a Bacillus strain. However, saidhost cell may also be a eukaryotic cell, preferably a yeast cell or afungal cell.

[0057] Finally this aspect of the present invention also comprises aprocess for the preparation of canthaxanthin by culturing saidrecombinant cell of the invention containing β-carotene in a culturemedium under suitable culture conditions whereby said β-caroteneβ4-oxygenase is expressed by said cell and catalyzes the conversion ofβ-carotene to produce echinenone and through further catalysis toproduce canthaxanthin, and isolating the canthaxanthin from such cellsor the culture medium.

[0058] It is contemplated, and in fact preferred, that theaforementioned DNA sequences, crtE_(E396), crtW_(E396) and crtZ_(E396),which terms refer to the above-described genes of microorganism E-396encompassed by the invention herein described, are incorporated,especially crtW_(E396) and crtZ_(E396), with selected DNA sequences fromFlavobacterium sp. R1534 into a polynucleotide of the invention wherebytwo or more of said DNA sequences which encode enzymes catalyzingcontiguious steps in the process shown in FIGS. 1 and 28 are containedin said polynucleotide, said polynucleotide being substantially free ofother polynucleotides of microorganism E-396 and Flavobacterium sp.R1534, to obtain advantageous production of the carotenoidscanthaxanthin, zeaxanthin, astaxanthin and adornixanthin.

[0059] Thus, one embodiment of the present invention is a process forthe preparation of zeaxanthin which process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to zeaxanthin, said recombinant cell comprisinga host cell transformed by an expression vector comprising a regulatorysequence and a polynucleotide containing DNA sequences which encode saidenzymes, as follows:

[0060] a) a DNA sequence which encodes the GGPP synthase ofFlavobacterium sp. R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence whichis substantially homologous,

[0061] b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

[0062] c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

[0063] d) a DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence whichis substantially homologous,

[0064] e) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 34) or a DNA sequencewhich is substantially homologous; and isolating the zeaxanthin fromsuch cells or the culture medium.

[0065] The above-described polynucleotide encodes enzymes which catalyzethe conversion of farnesyl pyrophosphate and isopentyl pyrophosphate tozeaxanthin. It is preferred that this embodiment of the inventionutilize a polynucleotide containing crtE, crtB, crtI, crtY, andcrtZ_(E396).

[0066] It is especially preferred that for this embodiment of theinvention:

[0067] a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQID NO: 2),

[0068] b) the prephytoene synthase has the amino acid sequence of FIG. 9(SEQ ID NO: 3),

[0069] c) the phytoene desaturase has the amino acid sequence of FIG. 10(SEQ ID NO: 4),

[0070] d) the lycopene cyclase has the amino acid sequence of FIG. 11(SEQ ID NO: 5), and

[0071] e) the β-carotene hydroxylase has the amino acid sequence of FIG.34.

[0072] It is most preferred that for this embodiment of the invention:

[0073] a) the DNA sequence encoding the GGPP synthase comprises bases2521-3408 of FIG. 7 (SEQ ID NO: 1),

[0074] b) the DNA sequence encoding the prephytoene synthase comprisesbases 4316-3405 of FIG. 7 (SEQ ID NO: 1),

[0075] c) the DNA sequence encoding the phytoene desaturase comprisesbases 4313-5797 of FIG. 7 (SEQ ID NO: 1),

[0076] d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1), and

[0077] e) the DNA sequence encoding the β-carotene hydroxylase comprisesthe sequence of FIG. 33 (SEQ ID NO: 33).

[0078] A second embodiment of the invention is a process for thepreparation of canthaxanthin which process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to canthaxanthin, said recombinant cellcomprising a host cell transformed by an expression vector comprising aregulatory sequence and a polynucleotide containing DNA sequences whichencode said enzymes, as follows:

[0079] a) a DNA sequence which encodes the GGPP synthase ofFlavobacterium sp. R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence whichis substantially homologous,

[0080] b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

[0081] c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

[0082] d) a DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence whichis substantially homologous, and

[0083] e) a DNA sequence which encodes the β-carotene β4-oxygenase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32) or a DNA sequencewhich is substantially homologous; and isolating the canthaxanthin fromsuch cells or the culture medium.

[0084] The above-described polynucleotide encodes enzymes which catalyzethe conversion of farnesyl pyrophosphate and isopentyl pyrophosphate tocanthaxanthin. It is preferred that this embodiment of the inventionutilize a polynucleotide containing crtE, crtB, crtI, crtY, andcrtW_(E396).

[0085] It is especially preferred that for this embodiment of theinvention:

[0086] a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQID NO: 2),

[0087] b) the prephytoene synthase has the amino acid sequence of FIG. 9(SEQ ID NO: 3),

[0088] c) the phytoene desaturase has the amino acid sequence of FIG. 10(SEQ ID NO: 4),

[0089] d) the lycopene cyclase has the amino acid sequence of FIG. 11(SEQ ID NO: 5), and

[0090] e) the β-carotene β4-oxygenase has the amino acid sequence ofFIG. 32 (SEQ ID NO: 32).

[0091] For this embodiment of the invention, it is most preferred that:

[0092] a) the DNA sequence encoding the GGPP synthase comprises bases2521-3408 of FIG. 7 (SEQ ID NO: 1),

[0093] b) the DNA sequence encoding the prephytoene synthase comprisesbases 4316-3405 of FIG. 7 (SEQ ID NO: 1),

[0094] c) the DNA sequence encoding the phytoene desaturase comprisesbases 4313-5797 of FIG. 7 (SEQ ID NO: 1),

[0095] d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1), and

[0096] e) the DNA sequence encoding the β-carotene β4-oxygenasecomprises the sequence of FIG. 31.

[0097] A third embodiment of the invention is a process for thepreparation of astaxanthin and adonixanthin wherein said processcomprises culturing a recombinant cell containing farnesyl pyrophosphateand isopentyl pyrophosphate under culture conditions sufficient for theexpression of enzymes which catalyze the conversion of the farnesylpyrophosphate and isopentyl pyrophosphate to astaxanthin andadonixanthin, said recombinant cell comprising a host cell transformedby an expression vector comprising a regulatory sequence and apolynucleotide containing DNA sequences which encode said enzymes, asfollows:

[0098] a) a DNA sequence which encodes the GGPP synthase ofFlavobacterium sp. R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence whichis substantially homologous,

[0099] b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

[0100] c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

[0101] d) a DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence whichis substantially homologous,

[0102] e) a DNA sequence which encodes the β-carotene b4-oxygenase ofFlavobacterium sp. R1534 (crtW) or a DNA sequence which is substantiallyhomologous, and

[0103] f) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) or a DNA sequence which issubstantially homologous; and isolating the astaxanthin and adonixanthinfrom such cells or the culture medium.

[0104] The above-described polynucleotide encodes enzymes which catalyzethe conversion of farnesyl pyrophosphate and isopentyl pyrophosphate toastaxanthin and adonixanthin. It is preferred that this embodiment ofthe invention utilize a polynucleotide containing crtE, crtB, crtI,crtY, crtW, and crtZ_(E396) (SEQ ID NO: 34).

[0105] It is especially preferred that for this embodiment of theinvention:

[0106] a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQID NO: 2),

[0107] b) the prephytoene synthase has the amino acid sequence of FIG. 9(SEQ ID NO: 3),

[0108] c) the phytoene desaturase has the amino acid sequence of FIG. 10(SEQ ID NO: 4),

[0109] d) the lycopene cyclase has the amino acid sequence of FIG. 11(SEQ ID NO: 5),

[0110] e) the β-carotene β4-oxygenase has the amino acid sequence ofFIG. 25 (SEQ ID NO: 29), and

[0111] f) the β-carotene hydroxylase has the amino acid sequence of FIG.34 (SEQ ID NO: 34).

[0112] It is most preferred that for this embodiment of the invention:

[0113] a) the DNA sequence encoding the GGPP synthase comprises bases2521-3408 of FIG. 7 (SEQ ID NO: 1),

[0114] b) the DNA sequence encoding the prephytoene synthase comprisesbases 4316-3405 of FIG. 7 (SEQ ID NO: 1),

[0115] c) the DNA sequence encoding the phytoene desaturase comprisesbases 4313-5797 of FIG. 7 (SEQ ID NO: 1),

[0116] d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1),

[0117] e) the DNA sequence encoding the β-carotene β4-oxygenasecomprises the sequence of FIG. 25 (SEQ ID NO: 28), and

[0118] f) the DNA sequence encoding the β-carotene hydroxylase comprisesthe sequence of FIG. 33 (SEQ ID NO: 33).

[0119] A fourth embodiment of the invention is a process for thepreparation of astaxanthin and adonixanthin wherein said processcomprises culturing a recombinant cell containing farnesyl pyrophosphateand isopentyl pyrophosphate under culture conditions sufficient for theexpression of enzymes which catalyze the conversion of the farnesylpyrophosphate and isopentyl pyrophosphate to astaxanthin andadonixanthin, said recombinant cell comprising a host cell transformedby an expression vector comprising a regulatory sequence and apolynucleotide containing DNA sequences which encode said enzymes, asfollows:

[0120] a) a DNA sequence which encodes the GGPP synthase ofFlavobacterium sp. R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence whichis substantially homologous,

[0121] b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

[0122] c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

[0123] d) a DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence whichis substantially homologous,

[0124] e) a DNA sequence which encodes the β-carotene β4-oxygenase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32) or a DNA sequencewhich is substantially homologous, and

[0125] f) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 34) or a DNA sequencewhich is substantially homologous; and isolating the astaxanthin andadonixanthin from such cells or the culture medium.

[0126] The above-described polynucleotide encodes enzymes which catalyzethe conversion of farnesyl pyrophosphate and isopentyl pyrophosphate toastaxanthin and adonixanthin. It is preferred that this embodiment ofthe invention utilize a polynucleotide containing crtE, crtB, crtI,crtY, crtW_(E396), and crtZ_(E396).

[0127] It is especially preferred that for this embodiment of theinvention:

[0128] a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQID NO: 2),

[0129] b) the prephytoene synthase has the amino acid sequence of FIG. 9(SEQ ID NO: 3),

[0130] c) the phytoene desaturase has the amino acid sequence of FIG. 10(SEQ ID NO: 4),

[0131] d) the lycopene cyclase has the amino acid sequence of FIG. 11(SEQ ID NO: 5),

[0132] e) the β-carotene β4oxygenase has the amino acid sequence of FIG.32 (SEQ ID NO: 32), and

[0133] f) the β-carotene hydroxylase has the amino acid sequence of FIG.34 (SEQ ID NO: 34).

[0134] It is most preferred that for this embodiment of the invention:

[0135] a) the DNA sequence encoding the GGPP synthase comprises bases2521-3408 of FIG. 7 (SEQ ID NO: 1),

[0136] b) the DNA sequence encoding the prephytoene synthase comprisesbases 4316-3405 of FIG. 7 (SEQ ID NO: 1),

[0137] c) the DNA sequence encoding the phytoene desaturase comprisesbases 4313-5797 of FIG. 7 (SEQ ID NO: 1),

[0138] d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1),

[0139] e) the DNA sequence encoding the β-carotene β4-oxygenasecomprises the sequence of FIG. 31 (SEQ ID NO: 31), and

[0140] f) the DNA sequence encoding the E-carotene hydroxylase comprisesthe sequence of FIG. 33 (SEQ ID NO: 33).

[0141] A fifth embodiment of the present invention is a process for thepreparation of adonixanthin wherein said process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to adonixanthin, said recombinant cellcomprising a host cell transformed by an expression vector comprising aregulatory sequence and a polynucleotide containing DNA sequences whichencode said enzymes, as follows:

[0142] a) a DNA sequence which encodes the GGPP synthase ofmicroorganism E-396 (crtE_(E396)) (SEQ ID NO: 37) or a DNA sequencewhich is substantially homologous,

[0143] b) a DNA sequence which encodes the prephytoene synthase ofmicroorganism E-396 (crtB_(E396)) or a DNA sequence which issubstantially homologous,

[0144] c) a DNA sequence which encodes the phytoene desaturase ofmicroorganism E-396 (crtI_(E396)) or a DNA sequence which issubstantially homologous,

[0145] d) a DNA sequence which encodes the lycopene cyclase ofmicroorganism E-396 (crtY_(E396)) or a DNA sequence which issubstantially homologous,

[0146] e) a DNA sequence which encodes the b-carotene b4-oxygenase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32) or a DNA sequencewhich is substantially homologous, and

[0147] f) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 33) or a DNA sequencewhich is substantially homologous,

[0148] said host cell being substantially free of other polynucleotidesof microorganism E-396; and isolating the adonixanthin from such cellsor the culture medium.

[0149] The above-described polynucleotide encodes enzymes which catalyzethe conversion of farnesyl pyrophosphate and isopentyl pyrophosphate toadonixanthin. It is preferred that this embodiment of the inventionutilize a polynucleotide containing crtE_(E396), crtB_(E396),crtI_(E396), crtY_(E396), crtW_(E396), and crtZ_(E396). It has beenfound that the use of the above-described process of the inventionresults in a preferential production of adonixanthin in relation toastaxanthin and other carotenoids. The preferred polynucleotide isplasmid pE396CARcrtW-E whose construction is described in Example 9herein.

[0150] The present invention also comprises the polynucleotidesdescribed above for the various embodiments of the invention and avector comprising such a polynucleotide, preferably in the form of anexpression vector. Furthermore the present invention also comprises arecombinant cell wherein said cell is a host cell which is transformedby a polynucleotide of the invention or vector which contains such apolynucleotide. Host cells useful for the expression of heterologousgenes normally contain farnesyl pyrophosphate and isopentylpyrophosphate, which are used for other purposes within the cell.Preferably said host cell is a prokaryotic cell and more preferably saidhost cell is an E. coli or a Bacillus strain. However, said host cellmay also be a eukaryotic cell, preferably a yeast cell or a fungal cell.

[0151] Finally the present invention also comprises a process for thepreparation of a desired carotenoid by culturing a recombinant cell ofthe invention containing a starting material in a culture medium undersuitable culture conditions and isolating the desired carotenoid fromsuch cells or the culture medium wherein the cell utilizes thepolynucleotide of the invention which contains said DNA sequences toexpress the enzymes which catalyze the reactions necessary to producethe desired carotenoid from the starting material. Where an enzymecatalyzes two sequential steps and it is preferred to produce theproduct of the second step (such as producing astaxanthin preferentiallyto adonixanthin (see FIG. 28)), a higher copy number of the DNA sequenceencoding the enzyme may be used to further production of the product ofthe second of the two steps in comparison to the first product. Thepresent invention further comprises a process for the preparation of afood or feed composition which process comprises mixing a nutritionallyeffective amount of the carotenoid isolated from the aforementionedrecombinant cells or culture medium with said food or feed.

[0152] In this context it should be mentioned that the expression “a DNAsequence is substantially homologous” refers with respect to the crtEencoding DNA sequence to a DNA sequence which encodes an amino acidsequence which shows more than 45%, preferably more than 60% and morepreferably more than 75% and most preferably more than 90% identicalamino acids when compared to the amino acid sequence of crtE ofFlavobacterium sp. 1534 and is the amino acid sequence of a polypeptidewhich shows the same type of enzymatic activity as the enzyme encoded bycrtE of Flavobacterium sp. 1534. In analogy with respect to crtB thismeans more than 60%, preferably more than 70%, more preferably more than80% and most preferably more than 90%; with respect to crtI this meansmore than 70%, preferably more than 80% and most preferably more than90%; with respect to crtY this means 55%, preferably 70%, morepreferably 80% and most preferably 90%.

[0153] “DNA sequences which are substantially homologous” refer withrespect to the crtW_(E396) encoding DNA sequence to a DNA sequence whichencodes an amino acid sequence which shows more than 60%, preferablymore than 75% and most preferably more than 90% identical amino acidswhen compared to the amino acid sequence of crtW of the microorganism E396 (FERM BP-4283) and is the amino acid sequence of a polypeptide whichshows the same type of enzymatic activity as the enzyme encoded by crtWof the microorganism E 396. In analogy with respect to crtZ_(E396) thismeans more than 75%, preferable more than 80% and most preferably morethan 90%; with respect to crtE_(E396), crtB_(E396), crtI_(E396),crtY_(E396) and crtZ_(E396) this means more than 80%, preferably morethan 90% and most preferably 95%.

[0154] The expression “said polynucleotide being substantially free ofother polynucleotides of Flavobacterium sp. R1534” and “saidpolynucleotide being substantially free of other polynucleotides ofmicroorganism E-396” is meant to preclude the present invention fromencompassing the polynucleotides as they exist in Flavobacterium sp.R1534 or in microorganism E-396, themselves. The polynucleotides hereindescribed which are combinations of two or more DNA sequences ofFlavobacterium sp. R1534 and/or microorganism E-396 are alsosubstantially free of other polynucleotides of Flavobacterium sp. R1534and microorganism E-396 in any circumstance where a polynucleotidecontaining only a single such DNA sequence would be substantially freeof other polynucleotides of Flavobacterium sp. R1534 or microorganismE-396.

[0155] DNA sequences in form of genomic DNA, cDNA or synthetic DNA canbe prepared as known in the art [see e.g. Sambrook et al., MolecularCloning, Cold Spring Habor Laboratory Press 1989] or, e.g. asspecifically described in Examples 1, 2 or 7. In the context of thepresent invention it should be noted that all DNA sequences used for theprocess for production of carotenoids of the present invention encodingcrt-gene products can also be prepared as synthetic DNA sequencesaccording to known methods or in analogy to the method specificallydescribed for crtW in Example 7.

[0156] The cloning of the DNA-sequences of the present invention fromsuch genomic DNA can than be effected, e.g. by using the well knownpolymerase chain reaction (PCR) method. The principles of this methodare outlined e.g. in PCR Protocols: A guide to Methods and Applications,Academic Press, Inc. (1990). PCR is an in vitro method for producinglarge amounts of a specific DNA of defined length and sequence from amixture of different DNA-sequences. Thereby, PCR is based on theenzymatic amplification of the specific DNA fragment of interest whichis flanked by two oligonucleotide primers which are specific for thissequence and which hybridize to the opposite strand of the targetsequence. The primers are oriented with their 3′ ends pointing towardeach other. Repeated cycles of heat denaturation of the template,annealing of the primers to their complementary sequences and extensionof the annealed primers with a DNA polymerase result in theamplification of the segment between the PCR primers. Since theextension product of each primer can serve as a template for the other,each cycle essentially doubles the amount of the DNA fragment producedin the previous cycle.

[0157] By utilizing the thermostable Taq DNA polymerase, isolated fromthe thermophilic bacteria Thermus aquaticus, it has been possible toavoid denaturation of the polymerase which necessitated the addition ofenzyme after each heat denaturation step. This development has led tothe automation of PCR by a variety of simple temperature-cyclingdevices. In addition, the specificity of the amplification reaction isincreased by allowing the use of higher temperatures for primerannealing and extension. The increased specificity improves the overallyield of amplified products by minimizing the competition by non-targetfragments for enzyme and primers. In this way the specific sequence ofinterest is highly amplified and can be easily separated from thenon-specific sequences by methods known in the art, e.g. by separationon an agarose gel and cloned by methods known in the art using vectorsas described e.g. by Holten and Graham in Nucleic Acid Res. 19, 1156(1991), Kovalic et. al. in Nucleic Acid Res. 19, 4560 (1991), Marchuk etal. in Nucleic Acid Res. 19, 1154 (1991) or Mead et al. inBio/Technology 9,657-663 (1991).

[0158] The oligonucleotide primers used in the PCR procedure can beprepared as known in the art and described e.g. in Sambrook et al., s.a.

[0159] Amplified DNA-sequences can than be used to screen DNA librariesby methods known in the art (Sambrook et al., s.a.) or as specificallydescribed in Examples 1 and 2.

[0160] Once complete DNA-sequences of the present invention have beenobtained they can be used as a guideline to define new PCR primers forthe cloning of substantially homologous DNA sequences from othersources. In addition they and such homologous DNA sequences can beintegrated into vectors by methods known in the art and described, e.g.,in Sambrook et al. (s.a.) to express or overexpress the encodedpolypeptide(s) in appropriate host systems. The expression vector intowhich the polynucleotides of the invention are integrated is notcritical. Conventional expression vectors may be selected based upon thesize of the polynucleotide of the invention to be inserted into thevector and the host cell to be transformed by the vector. Suchconventional expression vectors contain a regulatory sequence for thesynthesis of mRNA derived from the polynucleotide of the invention beingexpressed and possible marker genes. Conventional regulatory sequencesgenerally contain, but are not limited to, one or more of the following:a signal sequence, an origin of replication, one or more marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

[0161] However, a man skilled in the art knows that also theDNA-sequences themselves can be used to transform the suitable hostsystems of the invention to get overexpression of the encodedpolypeptide. Appropriate host systems are for example Bacteria e.g. E.coli , Bacilli as, e.g. Bacillus subtilis or Flavobacter strains. E.coli, which could be used are E. coli K12 strains e.g. M15 [described asDZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974)], HB 101[ATCC No. 33694] or E. coli SG13009 [Gottesman et al., J. Bacteriol.148, 265-273 (1981)].

[0162] Suitable Flavobacter strains can be obtained from any of theculture collections known to the man skilled in the art and listed, e.g.in the journal “Industrial Property” (January 1994, pgs 29-40), like theAmerican Type Culture Collection (ATCC) or the Centralbureau voorSchimmelkultures (CBS) and are, e.g. Flavobacterium sp. R 1534 (ATCC No.21588, classified as unknown bacterium; or as CBS 519.67) or allFlavobacter strains listed as CBS 517.67 to CBS 521.67 and CBS 523.67 toCBS 525.67, especially R 1533 (which is CBS 523.67 or ATCC 21081,classified as unknown bacterium; see also U.S. Pat. No. 3,841,967).Further Flavobacter strains are also described in WO 91/03571. Suitableeukaryotic host systems are for example fungi, like Aspergilli, e.g.Aspergillus niger [ATCC 9142] or yeasts, like Saccharomyces, e.g.Saccharomyces cerevisiae or Pichia, like pastoris, all available fromATCC.

[0163] Suitable vectors which can be used for expression in E. coli arementioned, e.g., by Sambrook et al. [s.a.] or by Fiers et al. in Procd.8th Int. Biotechnology Symposium” [Soc. Franc. de Microbiol., Paris(Durand et al., eds.), pp. 680-697 (1988)] or by Bujard et al. inMethods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc. Vol.155, 416-433 (1987) and Stuber et al. in Immunological Methods, eds.Lefkovits and Pernis, Academic Press, Inc., Vol. IV, 121-152 (1990).Vectors which could be used for expression in Bacilli are known in theart and described, e.g., in EP 405 370, EP 635 572 Procd. Nat. Acad.Sci. USA 81, 439 (1984) by Yansura and Henner, Meth. Enzym. 185, 199-228(1990) or EP 207 459. Vectors which can be used for expression in fungiare known in the art and described e.g. in EP 420 358 and for yeast inEP 183 070, EP 183 071, EP 248 227, EP 263 311. Vectors which can beused for expression in Flavobacter are known in the art and described inthe Examples or, e.g. in Plasmid Technology, edt. by J. Grinsted and P.M. Bennett, Academic Press (1990).

[0164] Once such DNA-sequences have been expressed in an appropriatehost cell in a suitable medium, the carotenoids can be isolated eitherfrom the medium in the case they are secreted into the medium or fromthe host organism and, if necessary separated from other carotenoids ifpresent in case one specific carotenoid is desired by methods known inthe art (see e.g. Carotenoids Vol IA: Isolation and Analysis, G.Britton, S. Liaaen-Jensen, H. Pfander; 1995, Birkhäuser Verlag, Basel).

[0165] The carotenoids of the present invention can be used in a processfor the preparation of food or feeds. A man skilled in the art isfamiliar with such processes. Such compound foods or feeds can furthercomprise additives or components generally used for such purpose andknown in the state of the art.

[0166] After the invention has been described in general hereinbefore,the following examples are intended to illustrate details of theinvention, without thereby limiting it in any matter.

EXAMPLE 1

[0167] Materials and General Methods Used

[0168] Bacterial strains and plasmids: Flavobacterium sp. R1534 WT (ATCC21588) was the DNA source for the genes cloned. Partial genomiclibraries of Flavobacterium sp. R1534 WT DNA were constructed into thepBluescriptII+(KS) or (SK) vector (Stratagene, La Jolla, USA) andtransformed into E. coli XL-1 blue (Stratagene) or JM109.

[0169] Media and growth conditions: Transformed E. coli were grown inLuria broth (LB) at 37° C. with 100 mg Ampicillin (Amp)/ml forselection. Flavobacterium sp. R1534 WT was grown at 27° C. in mediumcontaining 1% glucose, 1% tryptone (Difco Laboratories), 1% yeastextract (Difco), 0.5% MgSO₄ 7H₂O and 3% NaCl.

[0170] Colony screening: Screening of the E. coli transformants was doneby PCR basically according to the method described by Zon et al. [Zon etal., BioTechniques 7,696-698 (1989)] using the following primers: Primer#7 5′-CCTGGATGACGTGCTGGAATATTCC-3′ (SEQ ID NO:38): Primer #85′-CAAGGCCCAGATCGCAGGCG-3′ (SEQ ID NO:39):

[0171] Genomic DNA: A 50 ml overnight culture of Flavobacterium sp.R1534 was centrifuged at 10,000 g for 10 minutes. The pellet was washedbriefly with 10 ml of lysis buffer (50 mM EDTA, 0.1M NaCl pH7.5),resuspended in 4 ml of the same buffer sumplemented with 10 mg oflysozyme and incubated at 37° C. for 15 minutes. After addition of 0.3ml of N-Lauroyl sarcosine (20%) the incubation at 37° C. was continuedfor another 15 minutes before the extraction of the DNA with phenol,phenol/chloroform and chloroform. The DNA was ethanol precipitated atroom temperature for 20 minutes in the presence of 0.3 M sodium acetate(pH 5.2), followed by centrifugation at 10,000 g for 15 minutes. Thepellet was rinsed with 70% ethanol, dried and resuspended in 1 ml of TE(10 mM Tris, 1 mM EDTA, pH 8.0).

[0172] All genomic DNA used in the southern blot analysis and cloningexperiments was dialysed against H₂O for 48 hours, using collodium bags(Sartorius, Germany), ethanol precipitated in the presence of 0.3 Msodium acetate and resuspended in H₂O.

[0173] Probe labelling: DNA probes were labeled with (a-³²P) dGTP(Amersham) by random-priming according to [Sambrook et al., s.a.].

[0174] Probes used to screen the mini-libraries: Probe 46F is a 119 bpfragment obtained by PCR using primer #7 (SEQ ID NO: 38) and #8 (SEQ IDNO: 39) and Flavobacterium sp. R1534 genomic DNA as template. This probewas proposed to be a fragment of the Flavobacterium sp. R1534 phytoenesynthase (crtB) gene, since it shows significant homology to thephytoene synthase genes from other species (e.g. E. uredovora, E.herbicola). Probe A is a BstXI-PstI fragment of 184 bp originating fromthe right arm of the insert of clone 85. Probe B is a 397 bp XhoI-NotIfragment obtained from the left end of the insert of clone 85. Probe Cis a 536 bp BgIII-PstI fragment from the right end of the insert ofclone 85. Probe D is a 376 bp KpnI-BstYI fragment isolated from theinsert of clone 59. The localization of the individual probes is shownin FIG. 6.

[0175] Oligonucleotide synthesis: The oligonucleotides used for PCRreactions or for sequencing were synthesized with an Applied Biosystems392 DNA synthesizer.

[0176] Southern blot analysis: For hybridization experimentsFlavobacterium sp. R1534 genomic DNA (3 mg) was digested with theappropiate restriction enzymes and electrophoresed on a 0.75% agarosegel. The transfer to Zeta-Probe blotting membranes (BIO-RAD), was doneas described [Sourthern, E. M., J. Mol. Biol. 98, 503 (1975)].Prehybridization and hybridization was in 7% SDS, 1% BSA (fraction V;Boehringer), 0.5M Na₂HPO₄, pH 7.2 at 65° C. After hybridization themembranes were washed twice for 5 minutes in 2×SSC, 1% SDS at roomtemperature and twice for 15 minutes in 0.1% SSC, 0.1% SDS at 65° C.

[0177] DNA sequence analysis: The sequence was determined by the dideoxychain termination technique [Sanger et al., Proc. Natl. Acad. Sci. USA74, 5463-5467 (1977)] using the Sequenase Kit (United StatesBiochemical). Both strands were completely sequenced and the sequenceanalyzed using the GCG sequence analysis software package (Version 8.0)by Genetics Computer, Inc. [Devereux et al., Nucleic Acids. Res. 12,387-395 (1984)].

[0178] Analysis of carotenoids: E. coli XL-1 or JM109 cells (200-400 ml)carrying different plasmid constructs were grown for the times indicatedin the text, usually 24 to 60 hours, in LB suplemented with 100 mgAmpicillin/ml, in shake flasks at 37° C. and 220 rpm.

[0179] The carotenoids present in the microorganisms were extracted withan adequate volume of acetone using a rotation homogenizer (Polytron,Kinematica AG, CH-Luzern). The homogenate was the filtered through thesintered glass of a suction filter into a round bottom flask. Thefiltrate was evaporated by means of a rotation evaporator at 50° C.using a water-jet vacuum. For the zeaxanthin detection the residue wasdissolved in n-hexane/acetone (86:14) before analysis with a normalphaseHPLC as described in [Weber, S. in Analytical Methods for Vitamins andCarotenoids in Feed, Keller, H. E. , Editor, 83-85 (1988)]. For thedetection of β-carotene and lycopene the evaporated extract wasdissolved in n-hexane/acetone (99:1) and analysed by HPLC as describedin [Hengartner et al., Helv. Chim. Acta 75, 1848-1865 (1992)].

EXAMPLE 2

[0180] Cloning of the Flavobacterium sp. R1534 Carotenoid BiosyntheticGenes

[0181] To identify and isolate DNA fragments carrying the genes of thecarotenoid biosynthesis pathway, we used the DNA fragment 46F (seemethods) to probe a Southern Blot carrying chromosomal DNA ofFlavobacterium sp. R1534 digested with different restriction enzymesFIG. 2. The 2.4 kb XhoI/PstI fragment hybridizing to the probe seemedthe most appropiate one to start with. Genomic Flavobacterium sp. R1534DNA was digested with XhoI/PstI and run on a 1% agarose gel. Accordingto a comigrating DNA marker, the region of about 2.4 kb was cut out ofthe gel and the DNA isolated. A XhoI/PstI mini library of Flavobacteriumsp. R1534 genomic DNA was constructed into XhoI-PstI sites ofpBluescriptIISK(+). One hundred E. coli XL1 transformants weresubsequently screened by PCR with primer #7 (SEQ ID NO: 38) and primer #8 (SEQ ID NO: 39), the same primers previously used to obtain the 119 bpfragment (46F). One positive transformant, named clone 85, was found.Sequencing of the insert revealed sequences not only homologous to thephytoene synthase (crtB) but also to the phytoene desaturase (crtI) ofboth Erwinia species herbicola and uredovora. Left and right handgenomic sequences of clone 85 were obtained by the same approach usingprobe A and probe B. Flavobacterium sp. R1534 genomic DNA was doubledigested with ClaI and Hind III and subjected to Southern analysis withprobe A and probe B. With probe A a ClaI/HindIII fragment of aprox. 1.8kb was identified (FIG. 3A), isolated and subcloned into theClaI/HindIII sites of pBluescriptIIKS (+). Screening of the E. coli XL1transformants with probe A gave 6 positive clones. The insert of one ofthese positives, clone 43-3, was sequenced and showed homology to theN-terminus of crtI genes and to the C-terminus of crtY genes of bothErwinia species mentioned above. With probe B an approx. 9.2 kbClaI/HindlIl fragment was detected (FIG. 3B), isolated and subclonedinto pBluescriptIIKS (+) .

[0182] A screening of the transformants gave one positive, clone 51.Sequencing of the 5′ and 3′ of the insert, revealed that only the regionclose to the HindIII site showed relevant homology to genes of thecarotenoid biosynthesis of the Erwinia species mentioned above (e.g.crtB gene and crtE gene). The sequence around the ClaI site showed nohomology to known genes of the carotenoid biosynthesis pathway. Based onthis information and to facilitate further sequencing and constructionwork, the 4.2 kb BamHI/HindIII fragment of clone 51 was subcloned intothe respective sites of pBluescriptIIKS(+) resulting in clone 2.Sequencing of the insert of this clone confirmed the presence of geneshomologous to Erwinia sp crtB and crtE genes. These genes were locatedwithin 1.8 kb from the HindIII site. The remaining 2.4 kb of this inserthad no homology to known carotenoid biosynthesis genes.

[0183] Additional genomic sequences downstream of the ClaI site weredetected using probe C to hybridize to Flavobacterium sp. R1534 genomicDNA digested with different restriction enzymes (see FIG. 4).

[0184] A SalI/HindIII fragment of 2.8 kb identified by Southern analysiswas isolated and subcloned into the HindIII/XhoI sites ofpBluescriptIIKS (+). Screening of the E. coli XL1 transformants withprobe A gave one positive clone named clone 59. The insert of this cloneconfirmed the sequence of clone 43-3 and contained in addition sequenceshomologous to the N-terminus of the crtY gene from other known lycopenecyclases. To obtain the putative missing crtZ gene a Sau3AI partialdigestion library of Flavobacterium sp. R1534 was constructed into theBamHI site of pBluescriptIIKS(+). Screening of this library with probe Dgave several positive clones. One transformant designated, clone 6a, hadan insert of 4.9 kb. Sequencing of the insert revealed besides thealready known sequences coding for crtB, crtI and crtY also the missingcrtZ gene. Clone 7 g was isolated from a mini library carrying BclI/SphIfragments of R1534 (FIG. 5) and screened with probe D. The insert sizeof clone 7 g is approx. 3 kb.

[0185] The six independent inserts of the clones described abovecovering approx. 14 kb of the Flavobacterium sp. R1534 genome arecompiled in FIG. 6.

[0186] The determined sequence spanning from the BamHI site (position 1)to base pair 8625 is shown FIG. 7.

[0187] Putative Protein Coding Regions of the Cloned R1534 Sequence

[0188] Computer analysis using the CodonPreference program of the GCGpackage, which recognizes protein coding regions by virtue of thesimilarity of their codon usage to a given codon frequency table,revealed eight open reading frames (ORFs) encoding putative proteins: apartial ORF from 1 to 1165 (ORF-5) (SEQ ID NO: 41) coding for apolypeptide larger than 41382 Da; an ORF coding for a polypeptide with amolecular weight of 40081 Da from 1180 to 2352 (ORF-1) (SEQ ID NO: 40);an ORF coding for a polypeptide with a molecular weight of 31331 Da from2521 to 3405 (crtE); an ORF coding for a polypeptide with a molecularweight of 32615 Da from 4316 to 3408 (crtB); an ORF coding for apolypeptide with a molecular weight of 54411 Da from 5797 to 4316(crtI); an ORF coding for a polypeptide with a molecular weight of 42368Da from 6942 to 5797 (crtY); an ORF coding for a polypeptide with amolecular weight of 19282 Da from 7448 to 6942 (crtZ); and an ORF codingfor a polypeptide with a molecular weight of 19368 Da from 8315 to 7770(ORF-16) (SEQ ID NO: 42); ORF-1 and crtE have the oppositetranscriptional orientation from the others (FIG. 6). The translationstart sites of the ORFs crtI, crtY and crtZ could clearly be determinedbased on the appropiately located sequences homologous to theShine/Delgano (S/D) [Shine and Dalgarno, Proc. Natl. Acad. Sci. USA 71,1342-1346 (1974)] consensus sequence AGG-6-9N-ATG (FIG. 10) and thehomology to the N-terminal sequences of the respective enzymes of E.herbicola and E. uredovora. The translation of the ORF crtB couldpotentially start from three closely spaced codons ATG (4316), ATG(4241) and ATG (4211). The first one, although not having the best S/Dsequence of the three, gives a translation product with the highesthomology to the N-terminus of the E. herbicola and E. uredovora crtBprotein, and is therefore the most likely translation start site. Thetranslation of ORF crtE could potentially start from five differentstart codons found within 150 bp: ATG (2389), ATG (2446), ATG (2473),ATG (2497) and ATG (2521). We believe that based on the followingobservations, the ATG (2521) is the most likely transcription start siteof crtE: this ATG start codon is preceeded by the best consensus S/Dsequence of all five putative start sites mentioned; and the putativeN-terminal amino acid sequence of the protein encoded has the highesthomology to the N-terminus of the crtE enzymes of E. herbicola and E.uredovora;

[0189] Characteristics of the Crt Translational Initiation Sites andGene Products

[0190] The translational start sites of the five carotenoid biosynthesisgenes are shown below and the possible ribosome binding sites areunderlined. The genes crtZ, crtY, crtI and crtB are grouped so tightlythat the TGA stop codon of the anterior gene overlaps the ATG of thefollowing gene. Only three of the five genes (crtI, crtY and crtZ) fitwith the consensus for optimal S/D sequences. The boxed TGA sequenceshows the stop condon of the anterior gene.

crtE (SEQ ID NO: 43)

crtB (SEQ ID NO: 44)

crtY (SEQ ID NO: 45)

crtI (SEQ ID NO: 46)

crtZ (SEQ ID NO: 47)

[0191] Amino Acid Sequences of Individual Crt Genes of Flavobacteriumsp. R1534

[0192] All five ORFs of Flavobacterium sp. R1534 having homology toknown carotenoid biosynthesis genes of other species are clustered inapprox. 5.2 kb of the sequence (FIG. 7) (SEQ ID NO: 1).

[0193] FFDP synthase (crtE)

[0194] The amino acid (aa) sequence of the geranylgeranyl pyrophosphatesynthase (crtE gene product) consists of 295 aa and is shown in FIG. 8(SEQ ID NO: 2). This enzyme condenses farnesyl pyrophosphate andisopentenyl pyrophosphate in a 1′-4.

[0195] Phytoene synthase (crtB)

[0196] This enzyme catalyzes two enzymatic steps. First it condenses ina head to head reaction two geranylgeranyl pyrophosphates (C20) to theC40 carotenoid prephytoene. Second it rearanges the cyclopropylring ofprephytoene to phytoene. The 303 aa encoded by the crtB gene ofFlavobacterium sp. R1534 is shown in FIG. 9 (SEQ ID NO: 3).

[0197] Phytoene desaturase (crtI)

[0198] The phytoene desaturase of Flavobacterium sp. R1534 consisting of494 aa, shown in FIG. 10 (SEQ ID NO: 4), performs like the crtI enzymeof E. herbicola and E. uredovora, four desaturation steps, convertingthe non-coloured carotenoid phytoene to the red coloured lycopene.

[0199] Lycopene cyclase (crtY)

[0200] The crtY gene product of Flavobacterium sp. R1534 is sufficientto introduce the b-ionone rings at both sides of lycopene to obtainβ-carotene. The lycopene cyclase of Flavobacterium sp. R1534 consists of382 aa (FIG. 11) (SEQ ID NO: 5).

[0201] β-carotene hydroxylase (crtZ)

[0202] The gene product of crtZ consisting of 169 aa (FIG. 12) (SEQ IDNO: 6) and hydroxylates β-carotene to the xanthophyll zeaxanthin.

[0203] Putative Enzymatic Functions of the ORF's (Orf-1 (SEQ ID NO: 40),Orf-5 (SEQ ID NO: 41) and Orf-16 (SEQ ID NO: 42))

[0204] The orf-1 (SEQ ID NO: 40) has at the aa level over 40% identityto acetoacetyl-CoA thiolases of different organisms (e.g. Candidatropicalis, human, rat). This gene is therefore most likely a putativeacetoacetyl-CoA thiolase (acetyl-CoA acetyltransferase), which condensestwo molecules of acetyl-CoA to Acetoacetyl-CoA. Condensation ofacetoacetyl-CoA with a third acetyl-CoA by the HMG-CoA synthase formsβ-hydroxy-β-methylglutaryl-CoA (HMG-CoA). This compound is part of themevalonate pathway which produces besides sterols also numerous kinds ofisoprenoids with diverse cellular functions. In bacteria and plants, theisoprenoid pathway is also able to synthesize some unique products likecarotenoids, growth regulators (e.g. in plants gibberellins and abcissicacid) and sencodary metabolites like phytoalexins [Riou et al., Gene148, 293-297 (1994)].

[0205] The orf-5 (SEQ ID NO: 41) has a low homology of approx. 30%, tothe amino acid sequence of polyketide synthases from differentstreptomyces (e.g. S. violaceoruber, S. cinnamonensis). These antibioticsynthesizing enzymes (polyketide synthases), have been classified intotwo groups. Type-I polyketide synthases are large multifunctionalproteins, whereas type-II polyketide synthases are multiproteincomplexes composed of several individual proteins involved in thesubreactions of the polyketide synthesis [Bibb, et al. Gene 142, 31-39(1994)].

[0206] The putative protein encoded by the orf-16 (SEQ ID NO: 42) has atthe aa level an identity of 42% when compared to the soluble hydrogenasesubunit of Anabaena cylindrica.

[0207] Functional Assignment of the ORF 's (crtE, crtB, crtI, crtY andcrtZ) to Enzymatic Activities of the Carotenoid Biosynthesis Pathway

[0208] The biochemical assignment of the gene products of the differentORF's were revealed by analyzing carotenoid accumulation in E. coli hoststrains that were transformed with deleted variants of theFlavobacterium sp. gene cluster and thus expressed not all of the crtgenes (FIG. 13).

[0209] Three different plasmid were constructed: pLyco, p59-2 and pZea4.Plasmid p59-2 was obtained by subcloning the HindIII/BamHI fragment ofclone 2 into the HindIII/BamHI sites of clone 59. p59-2 carries theORF's of the crtE, crtB, crtI and crtY gene and should lead to theproduction of β-carotene. pLyco was obtained by deleting the KpnI/KpnIfragment, coding for approx. one half (N-terminus) of the crtY gene,from the p59-2 plasmid. E. coli cells transformed with pLyco, andtherefore having a truncated non-functional crtY gene, should producelycopene, the precursor of β-carotene. pZea4 was constructed by ligationof the AscI-SpeI fragment of p59-2, containing the crtE, crtB, crtI andmost of the crtY gene with the AscI/XbaI fragment of clone 6a,containing the sequences to complete the crtY gene and the crtZ gene.pZea4 [for complete sequence see FIG. 24 (SEQ ID NO: 27); nucleotides 1to 683 result from pBluescriptIIKS(+), nucleotides 684 to 8961 fromFlavobacterium R1534 WT genome, nucleotides 8962 to 11233 frompBluescriptIIKS(+)] has therefore all five ORF's of the zeaxanthinbiosynthesis pathway. Plasmid pZea4 has been deposited on May 25, 1995at the DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(Germany) under accession No. DSM 10012. E. coli cells transformed withthis latter plasmid should therefore produce zeaxanthin. For thedetection of the carotenoid produced, transformants were grown for 48hours in shake flasks and then subjected to carotenoid analysis asdescribed in the methods section. FIG. 13 summarizes the differentinserts of the plasmids described above, and the main carotenoiddetected in the cells.

[0210] As expected the pLyco carrying E. coli cells produced lycopene,those carrying p59-2 produced β-carotene (all-E,9-Z,13-Z) and the cellshaving the pZea4 construct produced zeaxanthin. This confirms that allthe necessary genes of Flavobacterium sp. R1534 for the synthesis ofzeaxanthin or their precursors (phytoene, lycopene and β-carotene) werecloned.

EXAMPLE 3

[0211] Materials and Methods Used for Expression of CarotenoidSynthesizing Enzymes

[0212] Bacterial strains and plasmids: The vectors pBluescriptIIKS (+)or (−) (Stratagene, La Jolla, USA) and pUC18 [Vieira and Messing, Gene19, 259-268 (1982); Norrander et al., Gene 26, 101-106 (1983)] were usedfor cloning in different E. coli strains, like XL-1 blue (Stratagene),TG1 or JM109. In all B. subtilis transformations, strain 1012 was used.Plasmids pHP13 [Haima et al., Mol. Gen. Genet. 209, 335-342 (1987)] andp602/22 [LeGrice, S. F. J. in Gene Expression Technology, Goeddel, D.V., Editor, 201-214 (1990)] are Gram (+)/(−) shuttle vectors able toreplicate in B. subtilis and E. coli cells. Plasmid p205 contains thevegI promoter cloned into the SmaI site of pUC18. Plasmid pXI12 is anintegration vector for the constitutive expression of genes in B.subtilis [Haiker et al., in 7th Int. Symposium on the Genetics ofIndustrial Microorganisms, Jun. 26-Jul. 1, 1994. Montreal, Quebec,Canada (1994)]. Plasmid pBEST501 [ltaya et al., Nucleic Acids Res. 17(11), 4410 (1989)] contains the neomycin resistance gene cassetteoriginating from the plasmid pUB110 (GenBank entry: M19465) of S. aureus[McKenzie et al., Plasmid 15, 93-103 (1986); McKenzie et al., Plasmid17, 83-84 (1987)]. This neomycin gene has been shown to work as aselection marker when present in a single copy in the genome of B.subtilis . Plasmid pC194 (ATCC 37034)(GenBank entry: L08860) originatesfrom S. aureuis [Horinouchi and Weisblaum, J. Bacteriol. 150, 815-825(1982)] and contains the chloramphenicol acetyltransferase gene.

[0213] Media and growth conditions: E. coli were grown in Luria broth(LB) at 37° C. with 100 mg Ampicillin (Amp)/ml for selection. B.subtilis cells were grown in VY-medium supplemented with eithererythromycin (1 mg/ml), neomycin (5-180 mg/ml) or chloramphenicol (10-80mg/ml).

[0214] Transformation: E. coli transformations were done byelectroporation using the Gene-pulser device of BIO-RAD (Hercules,Calif., USA) with the following parameters (200 W, 250 mFD, 2.5V). B.subtilis transformations were done basically according to the standardprocedure method 2.8 described by [Cutting and Vander Horn in MolecularBiological Methods for Bacillus, Harwood, C. R. and Cutting, S. M.,Editor, John Wiley & Sons: Chichester, England. 61-74 (1990)].

[0215] Colony screening: Bacterial colony screening was done asdescribed by [Zon et al., s.a.].

[0216] Oligonucleotide synthesis: The oligonucleotides used for PCRreactions or for sequencing were synthesized with an Applied Biosystems392 DNA synthesizer.

[0217] PCR reactions: The PCR reactions were performed using either theUlTma DNA polymerase (Perkin Elmer Cetus) or the Pfu Vent polymerase(New England Biolabs) according to the manufacturers instructions. Atypical 50 ml PCR reaction contained: 100 ng of template DNA, 10 pM ofeach of the primers, all four dNTP's (final conc. 300 mM), MgCl₂ (whenUlTma polymerase was used; final conc. 2 mM), 1×UlTma reaction buffer or1×Pfu buffer (supplied by the manufacturer). All components of thereaction with the exception of the DNA polymerase were incubated at 95°C. for 2 min. followed by the cycles indicated in the respective section(see below). In all reactions a hot start was made, by adding thepolymerase in the first round of the cycle during the 72° C. elongationstep. At the end of the PCR reaction an aliquot was analysed on 1%agarose gel, before extracting once with phenol/chloroform. Theamplified fragment in the aqueous phase was precipitated with 1/10 of a3M NaAcetate solution and two volumes of Ethanol. After centrifugationfor 5 min. at 12000 rpm, the pellet was resuspended in an adequatevolume of H₂O, typically 40 ml, before digestion with the indicatedrestriction enzymes was performed. After the digestion the mixture wasseparated on a 1% low melting point agarose. The PCR product of theexpected size were excised from the agarose and purified using the glassbeads method (GENECLEAN KIT, Bio 101, Vista Calif., USA) when thefragments were above 400 bp or directly spun out of the gel when thefragments were shorter than 400 bp, as described by [Heery et al., TIBS6 (6), 173 (1990)].

[0218] Oligos Used for Gene Amplification and Site Directed Mutagenesis

[0219] All PCR reactions performed to allow the construction of thedifferent plasmids are described below. All the primers used aresummarized in FIG. 14.

[0220] Primers #100 (SEQ ID NO: 7) and #101 (SEQ ID NO: 8) were used ina PCR reaction to amplify the complete crtE gene having a SpeIrestriction site and an artificial ribosomal binding site (RBS) upstreamof the transcription start site of this gene. At the 3′ end of theamplified fragment, two unique restriction sites were introduced, anAvrII and a SmaI site, to facilitate the further cloning steps. The PCRreaction was done with UlTma polymerase using the following conditionsfor the amplification: 5 cycles with the profile: 95° C., 1 min./60° C.,45 sec./72° C., 1 min. and 20 cycles with the profile: 95° C., 1min./72° C., 1 min. Plasmid pBIIKS(+)-clone2 served as template DNA. Thefinal PCR product was digested with SpeI and SmaI and isolated using theGENECLEAN KIT. The size of the fragment was approx. 910 bp.

[0221] Primers #104 (SEQ ID NO: 9) and #105 (SEQ ID NO: 10) were used ina PCR reaction to amplify the crtZ gene from the translation start tillthe SalI restriction site, located in the coding sequence of this gene.At the 5′ end of the crtZ gene an EcoRI, a synthetic RBS and a NdeI sitewas introduced. The PCR conditions were as described above. PlasmidpBIIKS(+)-clone 6a served as template DNA and the final PCR product wasdigested with EcoRI and SalI. Isolation of the fragment of approx. 480bp was done with the GENECLEAN KIT.

[0222] Primers MUT1 (SEQ ID NO: 11) and MUT5 (SEQ ID NO: 14) were usedto amplify the complete crtY gene. At the 5′ end, the last 23nucleotides of the crtZ gene including the SalI site are present,followed by an artificial RBS preceding the translation start site ofthe crtY gene. The artificial RBS created includes a PmlI restrictionsite. The 3′ end of the amplified fragment contains 22 nucleotides ofthe crtI gene, preceded by a newly created artificial RBS which containsa MunI restriction site. The conditions used for the PCR reaction wereas described above using the following cycling profile: 5 rounds of 95°C., 45 sec./60° C., 45 sec./72° C., 75 sec. followed by 22 cycles withthe profile: 95° C., 45 sec./66° C., 45 sec./72° C., 75 sec. PlasmidpXI12-ZYIB-EINV4 served as template for the Pfu Vent polymerase. The PCRproduct of 1225 bp was made blunt and cloned into the SmaI site ofpUC18, using the Sure-Clone Kit (Pharmacia) according to themanufacturer.

[0223] Primers MUT2 (SEQ ID NO: 15) and MUT6 (SEQ ID NO: 15) were usedto amplify the complete crtI gene. At the 5′ the last 23 nucleotides ofthe crtY gene are present, followed by an artificial RBS which precedesthe translation start site of the crtI gene. The new RBS created,includes a MunI restriction site. The 3′ end of the amplified fragmentcontains the artificial RBS upstream of the crtB gene including a BamHIrestriction site. The conditions used for the PCR reaction werebasically as described above including the following cycling profile: 5rounds of 95° C., 30 sec./60° C., 30 sec./72° C., 75 sec., followed by25 cycles with the profile: 95° C., 30 sec./66° C., 30 sec./72° C., 75sec. Plasmid pXI12-ZYIB-EINV4 served as template for the Pfu Ventpolymerase. For the further cloning steps the PCR product of 1541 bp wasdigested with MunI and BamHI.

[0224] Primers MUT3 (SEQ ID NO: 13) and CAR17 (SEQ ID NO: 16) were usedto amplify the N-terminus of the crtB gene. At the 5′ the last 28nucleotides of the crtI gene are present followed by an artificial RBS,preceding the translation start site of the crtB gene. This new createdRBS, includes a BamHI restriction site. The amplified fragment, namedPCR-F contains also the HindIII restriction site located at theN-terminus of the crtB gene. The conditions used for the PCR reactionwere as described elsewhere in the text, including the following cyclingprofile: 5 rounds of 95° C., 30 sec./58° C., 30 sec./72° C., 20 sec.followed by 25 cycles with the profile; 95° C., 30 sec./60° C., 30sec./72° C., 20 sec. Plasmid pXI12-ZYIB-EINV4 for the Pfu Ventpolymerase. The PCR product of approx. 160 bp was digested with BamHIand HindIII.

[0225] Oligos Used to Amplify the Chloramphenicol Resistance Gene (Cat)

[0226] Primers CAT3 (SEQ ID NO: 17) and CAT4 (SEQ ID NO: 18) were usedto amplify the chloramphenicol resistance gene of pC194 (ATCC 37034)[Horinouchi and Weisblum, s.a.] a R-plasmid found in S. aureus. Theconditions used for the PCR reaction were as described previouslyincluding the following cycling profile: 5 rounds of 95° C., 60 sec./50°C., 60 sec./72° C., 2 min. followed by 20 cycles with the profile: 95°C., 60 sec./60° C., 60 sec./72° C., 2 min.. Plasmid pC198 served astemplate for the Pfu Vent polymerase. The PCR product of approx. 1050 bpwas digested with EcoRI and AatII.

[0227] Oligos used to generate linkers: Linkers were obtained by adding90 ng of each of the two corresponding primers into an Eppendorf tube.The mixture was dried in a speed vac and the pellet resuspended in1×Ligation buffer (Boehringer, Mannheim, Germany). The solution wasincubated at 50° C. for 3 min. before cooling down to RT, to allow theprimers to hybridize properly. The linker were now ready to be ligatedinto the appropriate sites. All the oligos used to generate linkers areshown in FIG. 15.

[0228] Primers CS1 (SEQ ID NO: 19) and CS2 (SEQ ID NO: 20) were used toform a linker containing the following restrictions sites HindIII,AflII, ScaI, XbaI, PmeI and EcoRI.

[0229] Primers MUT7 (SEQ ID NO: 21) and MUT8 (SEQ ID NO: 22) were usedto form a linker containing the restriction sites SalI, AvrII, PmlI,MluI, MunI, BamHI, SphI and HindIII.

[0230] Primers MUT9 (SEQ ID NO: 23) and MUT10 (SEQ ID NO: 24) were usedto introduce an artificial RBS upstream of crtY.

[0231] Primers MUT11 (SEQ ID NO: 25) and MUT12 (SEQ ID NO: 26) were usedto introduce an artificial RBS upstream of crtE.

[0232] Isolation of RNA: Total RNA was prepared from log phase growingB. subtilis according to the method described by [Maes and Messens,Nucleic Acids Res. 20 (16), 4374 (1992)].

[0233] Northern blot analysis: For hybridization experiments up to 30 mgof B. subtilis RNA was electrophoreses on a 1% agarose gel made up in1×MOPS and 0.66 M formaldehyde. Transfer to Zeta-Probe blottingmembranes (BIO-RAD), UV cross-linking, pre-hybridization andhybridization was done as described elsewhere in [Farrell, J. R. E., RNAMethodologies. A laboratory Guide for isolation and characterization.San Diego, USA: Academic Press (1993)]. The washing conditions usedwere: 2×20 min. in 2×SSPE/0.1% SDS followed by 1×20 min. in 0.1%SSPE/0.1% SDS at 65° C. Northern blots were then analyzed either by aPhosphorimager (Molecular Dynamics) or by autoradiography on X-ray filmsfrom Kodak.

[0234] Isolation of genomic DNA: B. subtilis genomic DNA was isolatedfrom 25 ml overnight cultures according to the standard procedure method2.6 described by [13].

[0235] Southern blot analysis: For hybridization experiments B. subtilisgenomic DNA (3 mg) was digested with the appropriate restriction enzymesand electrophoresed on a 0.75% agarose gel. The transfer to Zeta-Probeblotting membranes (BIO-RAD), was done as described [Southern, E. M.,s.a.]. Prehybridization and hybridization was in 7%SDS, 1% BSA (fractionV; Boehringer), 0.5M Na₂HPO₄, pH 7.2 at 65° C. After hybridization themembranes were washed twice for 5 min. in 2×SSC, 1% SDS at roomtemperature and twice for 15 min. in 0.1% SSC, 0.1% SDS at 65° C.Southern blots were then analyzed either by a Phosphorimager (MolecularDynamics) or by autoradiography on X-ray films from Kodak.

[0236] DNA sequence analysis: The sequence was determined by the dideoxychain termination technique [Sanger et al., s.a.] using the SequenaseKit Version 1.0 (United States Biochemical). Sequence analysis were doneusing the GCG sequence analysis software package (Version 8.0) byGenetics Computer, Inc. [Devereux et al., s.a.].

[0237] Gene amplification in B. subtilis: To amplify the copy number ofthe SFCO in B. subtilis transformants, a single colony was inoculated in15 ml VY-medium supplemented with 1.5% glucose and 0.02 mgchloramphenicol or neomycin/ml, dependend on the antibiotic resistancegene present in the amplifiable structure (see results and discussion).The next day 750 ml of this culture were used to inoculate 13 mlVY-medium containing 1.5% glucose supplemented with (60, 80, 120 and 150mg/ml) for the cat resistant mutants, or 160 mg/ml and 180 mg/ml for theneomycin resistant mutants). The cultures were grown overnight and thenext day 50 ml of different dilutions (1:20, 1:400, 1:8000, 1:160′000)were plated on VY agar plates with the appropriate antibioticconcentration. Large single colonies were then further analyzed todetermine the number of copies and the amount of carotenoids produced.

[0238] Analysis of carotenoids: E. coli or B. subtilis transformants(200-400 ml) were grown for the times indicated in the text, usually 24to 72 hours, in LB-medium or VY-medium, respectively, supplemented withantibiotics, in shake flasks at 37° C. and 220 rpm.

[0239] The carotenoids produced by the microorganisms were extractedwith an adequate volume of acetone using a rotation homogenizer(Polytron, Kinematica AG, CH-Luzern). The homogenate was the filteredthrough the sintered glass of a suction filter into a round bottomflask. The filtrate was evaporated by means of a rotation evaporator at50° C. using a water-jet vacuum. For the zeaxanthin detection theresidue was dissolved in n-hexane/acetone (86:14) before analysis with anormalphase HPLC as described in [Weber, S., s.a.]. For the detection ofβ-carotene and lycopene the evaporated extract was dissolved inn-hexane/acetone (99:1) and analysed by HPLC as described in Hengartneret al., s.a.].

EXAMPLE 4

[0240] Carotenoid Production in E. coli

[0241] The biochemical assignment of the gene products of the differentopen reading frames (ORF's) of the carotenoid biosynthesis cluster ofFlavobacterium sp. were revealed by analyzing the carotenoidaccumulation in E. coli host strains, transformed with plasmids carryingdeletions of the Flavobacterium sp. gene cluster, and thus lacking someof the crt gene products. Similar functional assays in E. coli have beendescribed by other authors [Misawa et al., s.a.; Perry et al., J.Bacteriol., 168, 607-612 (1986); Hundle, et al., Molecular and GeneralGenetics 254 (4), 406-416 (1994)]. Three different plasmid pLyco,pBIIKS(+)-clone59-2 and pZea4 were constructed from the three genomicisolates pBIIKS(+)-clone2, pBIIKS(+)-clone59 and pBIIKS(+)-clone6a (seeFIG. 16).

[0242] Plasmid pBIIKS(+)-clone59-2 was obtained by subcloning theHindIII/BamHI fragment of pBIIKS(+)-clone 2 into the HindIII/BamHI sitesof pBIIKS(+)-clone59. The resulting plasmid pBIIKS(+)-clone59-2 carriesthe complete ORF's of the crtE, crtB, crtI and crtY gene and should leadto the production of β-carotene. pLyco was obtained by deleting theKpnI/KpnI fragment, coding for approx. one half (N-terminus) of the crtYgene, from the plasmid pBIIKS(+)-clone59-2. E. coli cells transformedwith pLyco, and therefore having a truncated non-functional crtY gene,should produce lycopene, the precursor of β-carotene. pZea4 wasconstructed by ligation of the AscI-SpeI fragment ofpBIIKS(+)-clone59-2, containing the crtE, crtB, crtI and most of thecrtY gene with the AscI/XbaI fragment of clone 6a, containing the crtZgene and sequences to complete the truncated crtY gene mentioned above.pZea4 has therefore all five ORF's of the zeaxanthin biosynthesispathway. E. coli cells transformed with this latter plasmid shouldtherefore produce zeaxanthin. For the detection of the carotenoidproduced, transformants were grown for 43 hours in shake flasks and thensubjected to carotenoid analysis as described in the methods section.FIG. 16 summarizes the construction of the plasmids described above.

[0243] As expected the pLyco carrying E. coli cells produced lycopene,those carrying pBIIKS(+)-clone59-2 produced β-carotene (all-E,9-Z,13-Z)and the cells having the pZea4 construct produced zeaxanthin. Thisconfirms that we have cloned all the necessary genes of Flavobacteriumsp. R1534 for the synthesis of zeaxanthin or their precursors (phytoene,lycopene and β-carotene). The production levels obtained are shown intable 1. TABLE 1 Carotenoid content of E. coli transformants, carryingthe plasmids pLyco, pBIIKS(+)-clone59-2 and pZea4, after 43 hours ofculture in shake flasks. The values indicated show the carotenoidcontent in % of the total dry cell mass (200 ml). ND = not detectable.plasmid host zeaxanthin β-χαρoτενε lycopene pLyco E. coli JM109 ND ND0.05% pBIIKS(+)- ″ ND 0.03% ND clone59-2 pZea4 ″ 0.033% 0.0009% ND

EXAMPLES 5

[0244] Carotenoid Production in B. subtilis

[0245] In a first approach to produce carotenoids in B. subtilis , wecloned the carotenoid biosynthesis genes of Flavobacterium into the Gram(+)/(−) shuttle vectors p602/22, a derivative of p602/20 [LeGrice, S. F.J., s.a.]. The assembling of the final construct p602-CARVEG-E, beginswith a triple ligation of fragments PvuII-AvrII of pZea4(del654-3028)and the AvrII-EcoRI fragment from plasmid pBIIKS(+)-clone6a, into theEcoRI and ScaI sites of the vector p602/22. The plasmidpZea4(del654-3028) had been obtained by digesting pZea4 with SacI andEspI. The protruding and recessed ends were made blunt with Klenowenzyme and religated. Construct pZea4(del654-3028) lacks most of thesequence upstream of crtE gene, which are not needed for the carotenoidbiosynthesis. The plasmid p602-CAR has approx. 6.7 kb of genomicFlavobacterium R1534 DNA containing besides all five carotenoid genes(approx. 4.9 kb), additional genomic DNA of 1.2 kb, located upstream ofthe crtZ translation start site and further 200 bp, located upstream ofcrtE transcription start. The crtZ, crtY, crtI and crtB genes werecloned downstream of the P_(N25/0) promoter, a regulatable E. colibacteriophage T5 promoter derivative, fused to a lac operator element,which is functional in B. subtilis [LeGrice, S. F. J., s.a.]. It isobvious that in the p602CAR construct, the distance of over 1200 bpbetween the P_(N25/0) promoter and the transcription start site of crtZis not optimal and will be improved at a later stage. An outline of thep602CAR construction is shown in FIG. 17. To ensure transcription of thecrtE gene in B. subtilis , the vegI promoter [Moran et al., Mol. Gen.Genet. 186, 339-346 (1982); LeGrice et al., Mol. Gen. Genet. 204,229-236 (1986)] was introduced upstream of this gene, resulting in theplasmid construct p602-CARVEG-E. The vegI promoter, which originatesfrom siteI of the veg promoter complex described by [LeGrice et al.,s.a.] has been shown to be functional in E. coli [Moran et al., s.a.].To obtain this new construct, the plasmid p602CAR was digested with SalIand HindIII, and the fragment containing the complete crtE gene and mostof the crtB coding sequence, was subcloned into the XhoI and HindIIIsites of plasmid p205. The resulting plasmid p205CAR contains the crtEgene just downstream of the PvegI promoter. To reconstitute thecarotenoid gene cluster of Flavobacterium sp. The following three pieceswere isolated: PmeI/HindIII fragment of p205CAR, the HincII/XbaIfragment and the EcoRI/HindIII fragment of p602CAR and ligated into theEcoRI and XbaI sites of pBluescriptIIKS(+), resulting in the constructpBIIKS(+)-CARVEG-E. Isolation of the EcoRI-XbaI fragment of this latterplasmid and ligation into the EcoRI and XbaI sites of p602/22 gives aplasmid similar to p602CAR but having the crtE gene driven by the PvegIpromoter. All the construction steps to get the plasmid p602CARVEG-E areoutlined in FIG. 18. E. coli TG1 cells transformed with this plasmidsynthesized zeaxanthin. In contrast B. subtilis strain 1012 transformedwith the same constructs did not produce any carotenoids. Analysis ofseveral zeaxanthin negative B. subtilis transformants always revealed,that the transformed plasmids had undergone severe deletions. Thisinstability could be due to the large size of the constructs.

[0246] In order to obtain a stable construct in B. subtilis , thecarotenoid genes were cloned into the Gram (+)/(−) shuttle vector pHP13constructed by [Haima et al., s.a.]. The stability problems were thoughtto be omitted by 1) reducing the size of the cloned insert which carriesthe carotenoid genes and 2) reversing the orientation of the crtE geneand thus only requiring one promoter for the expression of all fivegenes, instead of two, like in the previous constructs. Furthermore, theability of cells transformed by such a plasmid carrying the syntheticFlavobacterium carotenoid operon (SFCO), to produce carotenoids, wouldanswer the question if a modular approach is feasible. FIG. 19summarizes all the construction steps and intermediate plasmids made toget the final construct pHP13-2PNZYIB-EINV. Briefly: To facilitate thefollowing constructions, a vector pHP13-2 was made, by introducing asynthetic linker obtained with primer CS1 (SEQ ID NO: 19) and CS2 (SEQID NO: 20), between the HindIII and EcoRI sites of the shuttle vectorpHP13. The intermediate construct pHP13-2CARVEG-E was constructed bysubcloning the AflII-XbaI fragment of p602CARVEG-E into the AflII andXbaI sites of pHP13-2. The next step consisted in the inversion of crtEgene, by removing XbaI and AvrII fragment containing the original crtEgene and replacing it with the XbaI-AvrII fragment of plasmidpBIIKS(+)-PCRRBScrtE. The resulting plasmid was namedpHP13-2CARZYIB-EINV and represented the first construction with afunctional SFCO. The intermediate construct pBIIKS(+)-PCRRBScrtEmentioned above, was obtained by digesting the PCR product generatedwith primers #100 (SEQ ID NO: 7) and #101 (SEQ ID NO: 8) with SpeI andSmaI and ligating into the SpeI and SmaI sites of pBluescriptIIKS(+). Inorder to get the crtZ transcription start close to the promoterP_(N25/0) a triple ligation was done with the BamHI-SalI fragment ofpHP13-2CARZYIB-EINV (contains four of the five carotenoid genes), theBamHI-EcoRI fragment of the same plasmid containing the P_(N25/0)promoter and the EcoRI-SalI fragment of pBIIKS(+)-PCRRBScrtZ, havingmost of the crtZ gene preceded by a synthetic RBS. The aforementionedplasmid pBIISK(+)-PCRRBScrtZ was obtained by digesting the PCR productamplified with primers #104 (SEQ ID NO: 9) and #105 (SEQ ID NO: 10) withEcoRI and SalI and ligating into the EcoRI and SalI sites ofpBluescriptIISK(+). In the resulting vector pHP13-2PN25ZYIB-EINV, theSFCO is driven by the bacteriophage T5 promoter P_(N25/0,) which shouldbe constitutively expressed, due to the absence of a functional lacrepressor in the construct [Peschke and Beuk, J. Mol. Biol. 186, 547-555(1985)]. E. coli TG1 cells transformed with this construct producedzeaxanthin. Nevertheless, when this plasmid was transformed into B.subtilis , no carotenoid production could be detected. Analysis of theplasmids of these transformants showed severe deletions, pointingtowards instability problems, similar to the observations made with theaforementioned plasmids.

EXAMPLES 6

[0247] Chromosome Integration Constructs

[0248] Due to the instability observed with the previous constructs wedecided to integrate the carotenoid biosynthesis genes of Flavobacteriumsp. into the genome of B. subtilis using the integration/expressionvector pXI12. This vector allows the constitutive expression of wholeoperons after integration into the levan-sucrase gene (sacB) of the B.subtilis genome. The constitutive expression is driven by the vegIpromoter and results in medium level expression. The plasmidpXI12-ZYIB-EINV4 containing the synthetic Flavobacterium carotenoidoperon (SFCO) was constructed as follows: the NdeI-HincII fragment ofpBIISK(+)-PCRRBScrtZ was cloned into the NdeI and SmaI sites of pXI12and the resulting plasmid was named pXI12-PCRcrtZ. In the next step, theBstEII-PmeI fragment of pHP13-2PN25ZYIB-EINV was ligated to theBstEII-PmeI fragment of pXI12-PCRcrtZ (see FIG. 20). B. subtilistransformed with the resulting construct pXI12-ZYIB-EINV4 can integratethe CAR genes either via a Campbell type reaction or via a reciprocalrecombination. One transformant, BS1012::ZYIB-EINV4, having a reciprocalrecombination of the carotenoid biosynthesis genes into thelevan-sucrase gene was further analyzed (FIG. 21). Although this straindid not synthesize carotenoids, RNA analysis by Northern blots showedthe presence of specific polycistronic mRNA's of 5.4 kb and 4.2 kb whenhybridized to probe A (see FIG. 21, panel B). Whereas the larger mRNAhas the expected message size, the origin of the shorter mRNA wasunclear. Hybridization of the same Northern blot to probe B onlydetected the large mRNA fragment, pointing towards a prematuretermination of the transcription at the end of the crtB gene. Thepresence of a termination signal at this location would make sense,since in the original operon organisation in the Flavobacterium sp.R1534 genome, the crtE and the crtB genes are facing each other. Withthis constellation a transcription termination signal at the 5′ end ofcrtB would make sense, in order to avoid the synthesis of anti-sense RNAwhich could interfere with the mRNA transcript of the crtE gene. Sincethis region has been changed considerably with respect to the wild typesituation, the sequences constituting this terminator may also have beenaltered resulting in a “leaky” terminator. Western blot analysis usingantisera against the different crt enzymes of the carotenoid pathway,pointed towards the possibility that the ribosomal binding sites mightbe responsible for the lack of carotenoid synthesis. Out of the fivegenes introduced only the product of crtZ, the β-carotene hydroxylasewas detectable. This is the only gene preceded by a RBS site,originating from the pXI12 vector, known to be functional in B. subtilis. Base pairing interactions between a mRNA's Shine-Dalgarno sequence[Shine and Delagarno, s. a.] and the 16S rRNA, which permits theribosome to select the proper initiation site, have been proposed by[McLaughlin et al., J. Biol. Chem. 256,11283-11291 (1981)] to be muchmore stable in Gram-positive organisms (B. subtilis) than inGram-negative organisms (E. coli ). In order to obtain highly stablecomplexes we exchanged the RBS sites of the Gram-negative Flavobacteriumsp., preceding each of the genes crtY, crtI, crtB and crtE, withsynthetic RBS's which were designed complementary to the 3′ end of theB. subtilis 16S rRNA (see table 2). This exchange should allow aneffective translation initiation of the different carotenoid genes in B.subtilis . The strategy chosen to construct thispXI12-ZYIB-EINV4MUTRBS2C, containing all four altered sites issummarized in FIG. 20. In order to facilitate the further cloning stepsin pBluescriptIIKS(+), additional restriction sites were introducedusing the linker obtained with primer MUT7 and MUT8, cloned between theSalI and HindIII sites of said vector. The new resulting constructpBIIKS(+)-LINKER78 had the following restriction sites introduced:AvrII, PmlI, MulI, MunI, BamHI and SphI. The general approach chosen tocreate the synthetic RBS's upstream of the different carotenoid genes,was done using a combination of PCR based mutagenesis, where the geneswere reconstructed using defined primers carrying the modified RBSsites, or using synthetic linkers having such sequences. Reconstitutionof the RBS preceding the crtI and crtB genes was done by amplifying thecrtI gene with the primers MUT2 (SEQ ID NO: 12) and MUT6 (SEQ ID NO:15), which include the appropriate altered RBS sites. The PCR-I fragmentobtained was digested with MunI and BamHI and ligated into the MunI andBamHI sites of pBIIKS(+)-LINKER78. The resulting intermediate constructwas named pBIIKS(+)-LINKER78PCRI. Reconstitution of the RBS precedingthe crtB gene was done using a small PCR fragment obtained with primerMUT3 (SEQ ID NO: 13), carrying the altered RBS site upstream of crtB,and primer CAR17 (SEQ ID NO: 16). The amplified PCR-F fragment wasdigested with BamHI and HindIII and sub cloned into the BamHI andHindIII sites of pBIIKS(+)-LINKER78, resulting in the constructpBIIKS(+)-LINKER78PCRF. The PCR-I fragment was cut out ofpBIIKS(+)-LINKER78PCRI with BamHI and SapI and ligated into the BamHIand SapI sites of pBIIKS(+)-LINKER78PCRF. The resulting plasmidpBIIKS(+)-LINKER78PCRFI has the PCR-I fragment fused to the PCR-Ffragment. This construct was cut with SalI and PmlI and a syntheticlinker obtained by annealing of primer MUT9 (SEQ ID NO: 23) and MUT10(SEQ ID NO: 24) was introduced. This latter step was done to facilitatethe upcoming replacement of the original Flavobacterium RBS in the abovementioned construct. The resulting plasmid was namedpBIIKS(+)-LINKER78PCRFIA. Assembling of the synthetic RBS's precedingthe crtY and crtI genes was done by PCR, using primers MUT1 (SEQ ID NO:11) and MUT5 (SEQ ID NO: 14). The amplified fragment PCR-G was madeblunt end before cloning into the SmaI site of pUC18, resulting inconstruct pUC18-PCR-G. The next step was the cloning of the PCR-Gfragment between the PCR-A and PCR-I fragments. For this purpose thePCR-G was isolated from pUC18-PCR-G by digesting with MunI and PmlI andligated into the MunI and PmlI sites of pBIIKS(+)-LINKER78PCRFIA. Thisconstruct contains all four fragments, PCR-F, PCR-I, PCR-G and PCR-A,assembled adjacent to each other and containing three of the fourartificial RBS sites (crtY, crtI and crtB). The exchange of theFlavobacterium RBS's preceding the genes crtY, crtI and crtB bysynthetic ones, was done by replacing the HindIII-SalI fragment ofplasmid pXI12-ZYIB-EINV4 with the HindIII-SalI fragment of plasmidpBIIKS(+)-LINKER78PCRFIGA. The resulting plasmid pXI12-ZYIB-EINV4MUTRBSC was subsequently transformed into E. coli TG1 cells and B.subtilis 1012. The production of zeaxanthin by these cells confirmedthat the PCR amplified genes where functional. The B. subtilis strainobtained was named BS1012::SFCO1. The last Flavobacterium RBS to beexchanged was the one preceding the crtE gene. This was done using alinker obtained using primer MUT11 (SEQ ID NO: 25) and MUT12 (SEQ ID NO:26). The wild type RBS was removed from pXI12-ZYIB-EINV4MUTRBS with NdeIand SpeI and the above mentioned linker was inserted. In the constructpXI12-ZYIB-EINV4MUTRBS2C all Flavobacterium RBS's have been replaced bysynthetic RBS's of the consensus sequence AAAGGAGG-7-8N-ATG (see table2). E. coli TG1 cells transformed with this construct showed that alsothis last RBS replacement had not interferred TABLE 2 mRNA nucleotidesequence crtZ (SEQ ID NO: 48) AAAGGAGGGUUUCAUAUGAGC crtY (SEQ ID NO: 49)AAAGGAGGACACGUGAUGAGC crtI (SEQ ID NO: 50) AAAGGAGGCAAUUGAGAUGAGU crtB(SEQ ID NO: 51) AAAGGAGGAUCCAAUCAUGACC crtE (SEQ ID NO: 52)AAAGGAGGGUUUCUUAUGACG B. subtilis 16S rRNA (SEQ ID NO: 53)3′-UCUUUCCUCCACUAG E. coli 16S rRNA (SEQ ID NO: 54) 3′- AUUCCUCCACUAGTABLE 2: Nucleotide sequences of the synthetic ribosome binding sites inthe constructs pXI12-ZYIB3-EINV4MUTRBS2C, pXI12-ZYIB-EINV4MUTRBS2CCATand pXI12-ZYIB-EINV4 MUTRBS2CNEO. Nucleotides of the Shine-Dalgarnosequence preceding the individual carotenoid genes which arecomplementary to the 3′ ends of the 16S rRNA of B. subtilis are shown inbold. The 3′ ends of the 16S #rRNA of E. coli is also shown ascomparison. The underlined AUG is the translation start site of thementioned gene.

[0249] with the ability to produce zeaxanthin. All the regionscontaining the newly introduced synthetic RBS's were confirmed bysequencing. B. subtilis cells were transformed with plasmidpXI12-ZYIB-EINV4MUTRBS2 and one transformant having integrated the SFCOby reciprocal recombination, into the levan-sucrase gene of thechromosome, was selected. This strain was named BS1012::SFCO2. Analysisof the carotenoid production of this strain show that the amountszeaxanthin produced is approx. 40% of the zeaxanthin produced by E. colicells transformed with the plasmid used to get the B. subtilistransformant. Similar was the observation when comparing theBS1012::SFCO1 strain with its E. coli counter part (30%). Although theE. coli cells have 18 times more carotenoid genes, the carotenoidproduction is only a factor of 2-3 times higher. More drastic was thedifference observed in the carotenoid contents, between E. coli cellscarrying the pZea4 construct in about 200 copies and the E. colicarrying the plasmid pXI12-ZYIB-EINV4MUTRBS2C in 18 copies. The firsttransformant produced 48× more zeaxanthin than the latter one. Thisdifference seen can not only be attributed to the roughly 11 times morecarotenoid biosynthesis genes present in these transformants.Contributing to this difference is probably also the suboptimalperformance of the newly constructed SFCO, in which the overlappinggenes of the wild type Flavobacterium operon were separated to introducethe synthetic RBS's. This could have resulted in a lower translationefficiency of the rebuild synthetic operon (e.g. due to elimination ofputative translational coupling effects, present in the wild typeoperon).

[0250] In order to increase the carotenoid production, two newconstructs were made, pXI12-ZYIB-EINV4MUTRBS2CNEO and pXI12-ZYIB-EINV4MUTRBS2CCAT, which after the integration of the SFCO into thelevan-sucrase site of the chromosome, generate strains with anamplifiable structure as described by [Janniere et al., Gene 40, 47-55(1985)]. Plasmid pXI12-ZYIB-EINV4MUTRBS2CNEO has been deposited on May25, 1995 at the DSM-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (Germany) under accession No. DSM 10013. Suchamplifiable structures, when linked to a resistance marker (e.gchloramphenicol, neomycin, tetracycline), can be amplified to 20-50copies per chromosome. The amplifiable structure consist of the SFCO,the resistance gene and the pXI12 sequence, flanked by direct repeats ofthe sac-B 3′ gene (see FIG. 22). New strains having elevated numbers ofthe SFCO could now be obtained by selecting for transformants withincreased level of resistance to the antibiotic. To construct plasmidpXI12-ZYIB-EINV4MUTRBS2CNEO, the neomycin resistance gene was isolatedfrom plasmid pBEST501 with PstI and SmaI and subcloned into the PstI andEcoO1091 sites of the pUC18 vector. The resulting construct was namedpUC18-Neo. To get the final construct, the PmeI-AatII fragment ofplasmid pXI12-ZYIB-EINV4MUTRBS2C was replaced with the SmaI-AatIIfragment of pUC18-Neo, containing the neomycin resistance gene. PlasmidpXI12-ZYIB-EINV4MUTRBS2CCAT was obtained as follows: the chloramphenicolresistance gene of pC194 was isolated by PCR using the primer pair cat3(SEQ ID NO: 17) and cat4 (SEQ ID NO: 18). The fragment was digested withEcoRI and AatII and subcloned into the EcoRI and AatII sites of pUC18.The resulting plasmid was named pUC18-CAT. The final vector was obtainedby replacing the PmeI-AatIl fragment of pXI12-ZYIB-EINV4MUTRBS2C withthe EcoRI-AatII fragment of pUC18-CAT, carrying the chloramphenicolresistance gene. FIG. 23 summarizes the different steps to obtainaforementioned constructs. Both plasmids were transformed into B.subtilis strain 1012, and transformants resulting from a Campbell-typeintegration were selected. Two strains BS1012::SFCONEO1 andBS1012::SFCOCAT1 were chosen for further amplification. Individualcolonies of both strains were independently amplified by growing them indifferent concentrations of antibiotics as described in the methodssection. For the cat gene carrying strain, the chloramphenicolconcentrations were 60, 80, 120 and 150 mg/ml. For the neo gene carryingstrain, the neomycin concentrations were 160 and 180 mg/ml. In bothstrains only strains with minor amplifications of the SFCO's wereobtained. In daughter strains generated from strain BS1012::SFCONEO1,the resistance to higher neomycin concentrations correlated with theincrease in the number of SFCO's in the chromosome and with higherlevels of carotenoids produced by these cells. A different result wasobtained with daughter strains obtained from strain BS1012::SFCOCAT1. Inthese strains an increase up to 150 mg chloramphenicol/ml resulted, asexpected, in a higher number of SFCO copies in the chromosome.

EXAMPLE 7

[0251] Construction of CrtW Containing Plasmids and Use for CarotenoidProduction

[0252] Polymerase chain reaction based gene synthesis: The nucleotidesequence of the artificial crtW gene, encoding the β-caroteneβ-4-oxygenase of Alcaligenes strain PC-1, was obtained by backtranslating the amino acid sequence outlined in [Misawa, 1995], usingthe BackTranslate program of the GCG Wisconsin Sequence AnalysisPackage, Version 8.0 (Genetics Computer Group, Madison, Wis., USA) and acodon frequency reference table of E. coli (supplied by the BachTranslate Program). The synthetic gene consisting of 726 nucleotides wasconstructed basically according to the method described by [Ye, 1992].The sequence of the 12 oligonucleotides (crtW1- crtW12) required for thesynthesis are shown in FIG. 25 (SEQ ID NO: 28). Briefly, the longoligonucleotides were designed to have short overlaps of 15-20 bases,serving as primers for the extension of the oligonucleotides. After fourcycles a few copies of the full length gene should be present which isthen amplified by the two terminal oligonucleotides crtW15 (SEQ ID NO:55) and crtW26. The sequences for these two short oligonucleotides arefor the forward primer crtW15 (5′-TATATCTAGAcatatgTCCGGTCGTAAA CCGG-3′)and for the reverse primer crtW26 (SEQ ID NO: 56)(5′-TATAgaattccacgtgTCA AGCACGA CCACCGGTTTTAC G-3′), where the sequencesmatching the DNA templates are underlined. Small cap letters show theintroduced restriction sites (NdeI for the forward primer and EcoRI andPmlI for the reverse primer) for the latter cloning into the pALTER-Ex2expression vector.

[0253] Polymerase chain reaction: All twelve long oligonucleotides(crtW1-crtW12; 7 nM each) and both terminal primers (crtW15 and crtW26;0.1 mM each) were mixed and added to a PCR reaction mix containingExpand™ High Fidelity polymerase (Boehringer, Mannheim) (3.5 units) anddNTP's (100 mM each). The PCR reaction was run for 30 cycles with thefollowing profile: 94° C. for 1 min, 50° C. for 2 min and 72° C. for 3min. The PCR reaction was separated on a 1% agarose gel, and the band ofapprox. 700 bp was excised and purified using the glass beads method(Geneclean Kit, Bio101, Vista, Calif., USA). The fragment wassubsequently cloned into the SmaI site of plasmid pUC18, using theSure-Clone Kit (Pharmacia, Uppsala, Sweden). The sequence of theresulting crtW synthetic gene was verified by sequencing with theSequenase Kit Version 1.0 (United States Biochemical, Cleveland, Ohio,USA). The crtW gene constructed by this method was found to containminor errors, which were subsequently corrected by site-directedmutagenesis.

[0254] Construction of plasmids: Plasmid pBIIKS(+)-CARVEG-E (see alsoExample 5) (FIG. 26) contains the carotenoid biosynthesis genes (crtE,crtB, crtY, crtI and crtZ) of the Gram (−) bacterium Flavobacterium sp.strain R1534 WT (ATCC 21588) [Pasamontes, 1995 #732] cloned into amodified pBluescript II KS(+) vector (Stratagene, La Jolla, USA)carrying site I of the B. subtilis veg promoter [LeGrice, 1986 #806].This constitutive promoter has been shown to be functional in E. coli .Transformants of E. coli strain TG1 carrying plasmid pBIIKS(+)-CARVEG-Esynthesise zeaxanthin. Plasmid pALTER-Ex2-crtW was constructed bycloning the NdeI-EcoRI restricted fragment of the synthetic crtW geneinto the corresponding sites of plasmid pALTER-Ex2 (Promega, Madison,Wis.). Plasmid pALTER-Ex2 is a low copy plasmid with the p15a origin ofreplication, which allows it to be maintained with ColE1 vectors in thesame host. Plasmid pBIIKS-crtEBIYZW (FIG. 26) was obtained by cloningthe HindIII-PmlI fragment of pALTER-Ex2-crtW into the HindIII and theblunt end made MluI site obtained by a fill in reaction with Klenowenzyme, as described elsewhere in [Sambrook, 1989 #505]. Inactivation ofthe crtZ gene was done by deleting a 285 bp NsiI-NsiI fragment, followedby a fill in reaction and religation, resulting in plasmidpBIIKS-crtEBIY[DZ]W. Plasmid pBIIKS-crtEBIY[DZW] carrying thenon-functional genes crtW and crtZ, was constructed by digesting theplasmid pBIIKS-crtEBIY[DZ]W with NdeI and HpaI, and subsequent selfreligation of the plasmid after filling in the sites with Klenow enzyme.E. coli transformed with this plasmid had a yellow-orange colour due tothe accumulation of β-carotene. Plasmid pBIIKS-crtEBIYZ[DW] has atruncated crtW gene obtained by deleting the NdeI-HpaI fragment inplasmid pBIIKS-crtEBIYZW as outlined above. PlasmidspALTER-Ex2-crtEBIY[DZW] and pALTER-Ex2-crtEBIYZ[DW], were obtained byisolating the BamHI-XbaI fragment from pBIIKS-crtEBIY[DZW] andpBIIKS-crtEBIYZ[DW], respectively and cloning them into the BamHI andXbaI sites of pALTER-Ex2. The plasmid pBIIKS-crtW was constructed bydigesting pBIIKS-crtEBIYZW with NsiI and SacI, and self-religating theplasmid after recessing the DNA overhangs with Klenow enzyme. FIG. 27compiles the relevant inserts of all the plasmids used in this paper.

[0255] Carotenoid analysis: E. coli TG-1 transformants carrying thedifferent plasmid constructs were grown for 20 hours in Luria-Brothmedium supplemented with antibiotics (ampicillin 100 mg/ml, tetracyclin12.5 mg/ml) in shake flasks at 37° C. and 220 rpm. Carotenoids wereextracted from the cells with acetone. The acetone was removed in vacuoand the residue was re dissolved in toluene. The coloured solutions weresubjected to high-performance liquid chromatography (HPLC) analysiswhich was performed on a Hewlett-Packard series 1050 instrument. Thecarotenoids were separated on a silica column Nucleosil Si-100, 200×4mm, 3m. The solvent system included two solvents: hexane (A) andhexane/THF, 1:1 (B). A linear gradient was applied running from 13 to50% (B) within 15 minutes. The flow rate was 1.5 ml/min. Peaks weredetected at 450 nm by a photo diode array detector. The individualcarotenoid pigments were identified by their absorption spectra andtypical retention times as compared to reference samples of chemicallypure carotenoids, prepared by chemical synthesis and characterised byNMR, MS and UV-Spectra. HPLC analysis of the pigments isolated from E.coli cells transformed with plasmid pBIIKS-crtEBIYZW, carrying besidesthe carotenoid biosynthesis genes of Flavobacterium sp. strain R1534,also the crtW gene encoding the β-carotene ketolase of Alcaligenes PC-1[Misawa, 1995 #670] gave the following major peaks identified as:b-cryptoxanthin, astaxanthin, adonixanthin and zeaxanthin, based on theretention times and on the comparison of the absorbance spectra to givenreference samples of chemically pure carotenoids. The relative amount(area percent) of the accumulated pigment in the E. coli transformantcarrying pBIIKS-crtEBIYZW is shown in Table 3 [“CRX”: cryptoxanthin;“ASX”: astaxanthin; “ADX”: adonixanthin; “ZXN”: zeaxanthin; “ECM”:echinenone; “MECH”: 3-hydroxyechinenone, “CXN”: canthaxanthin]. The Σ ofthe peak areas of all identified carotenoids was defined as 100%.Numbers shown in Table 3 represent the average value of four independentcultures for each transformant. In contrast to the aforementionedresults, E. coli transformants carrying the same genes but on twoplasmids namely, pBIIKS-crtEBIYZ[DW] and pALTER-Ex2-crtW, showed adrastical drop in adonixanthin and a complete lack of astaxanthinpigments (Table 3), whereas the relative amount of zeaxanthin (%) hadincreased. Echinenone, hydroxyechinenone and canthaxanthin levelsremained unchanged compared to the transformants carrying all the crtgenes on the same plasmid (pBIIKS-crtEBIYZDW). PlasmidpBIIKS-crtEBIYZ[DW] is a high copy plasmid carrying the functional genesof crtE, crtB, crtY, crtI, crtZ of Flavobacterium sp. strain R1534 and atruncated, non-functional version of the crtW gene, whereas thefunctional copy of the crtW gene is located on the low copy plasmidpALTER-Ex2-crtW. To analyze the effect of overexpression of the crtWgene with respect to the crtZ gene, E. coli cells were co-transformedwith plasmid pBIIKS-crtW carrying the crtW gene on the high copy plasmidpBIIKS-crtW and the low copy construct pALTER-Ex2-crtEBIYZ[DW], encodingthe Flavobacterium crt genes. Pigment analysis of these transformants byHPLC monitored the presence of β-carotene, cryptoxanthin, astaxanthin,adonixanthin, zeaxanthin, 3-hydroxyechine-none and minute traces ofechinenone and canthaxanthin (Table 3).

[0256] Transformants harbouring the crtW gene on the low copy plasmidpALTER-Ex2-crtW and the genes crtE, crtB, crtY and crtI on the high copyplasmid pBIIKS-crtEBIY[DZW] expressed only minor amounts ofcanthaxanthin (6%) but high levels of echinenone (94%), whereas cellscarrying the crtW gene on the high copy plasmid pBIIKS-crtW and theother crt genes on the low copy construct pALTER-Ex2-crtEBIY[DZW], had78.6% and 21.4% of echinenone and canthaxanthin, respectively (Table 3).TABLE 3 plasmids CRX ASX ADX ZXN ECH HECH CXN pBIIKS-crtEBIYZW 1.1 2.044.2 52.4 <1 <1 <1 pBIIKS-crtEBIYZ[W] + 2.2 — 25.4 72.4 <1 <1 <1pALTER-Ex2-crtW pBIIKS-crtEBIY[Z]W — — — — 66.5 — 33.5pBIIKS-crtEBIY[ZW] + pBIIKS- — — — — 94 — 6 crtW

EXAMPLE 8

[0257] Selective Carotenoid Production by Using the CrtW and CrtZ Genesof the Gram Negative Bacterium E-396

[0258] In this section we describe E. coli transformants whichaccumulate only one (canthaxanthin) or two main carotenoids(astaxanthin, adonixanthin) and minor amounts of adonirubin, rather thanthe complex variety of carotenoids seen in most carotenoid producingbacteria [Yokoyama et al., Biosci. Biotechnol. Biochem. 58:1842-1844(1994)] and some of the E. coli transformants shown in Table 3. Theability to construct strains producing only one carotenoid is a majorstep towards a successful biotechnological carotenoid productionprocess. This increase in the accumulation of individual carotenoidsaccompanied by a decrease of the intermediates, was obtained byreplacing the crtZ of Flavobacterium R1534 and/or the synthetic crtWgene (see example 5) by their homologous genes originating from theastaxanthin producing Gram negative bacterium E-396 (FERM BP-4283)[Tsubokura et al., EP-application 0 635 576 A1]. Both genes, crtW_(E396)and crtZ_(E396), were isolated and used to construct new plasmids asoutlined below.

[0259] Isolation of a putative fragment of the crtW gene of strain E-396by the polymerase chain reaction: Based on protein sequence comparisonof the crtW enzymes of Agrobacterium aurantiacum, Alcaligenes PC-1(WO95/18220) [Misawa et al., J.Bacteriol. 177: 6575-6584 (1995)] andHaematococcus pluvialis [Kajiwara et al., Plant Mol. Biol. 29:343-352(1995)][Lotan et al., FEBS letters, 364:125-128 (1995)], two regionsnamed I and II, having high amino acid conservation and located approx.140 amino acids appart, were identified and chosen to design thedegenerate PCR primers shown below. The N-terminal peptide HDAMHG(region I) was used to design the two 17-mer degenerate primer sequencescrtW100 (SEQ ID NO: 57) and crtW101 (SEQ ID NO: 58): crtW100 (SEQ ID NO:57): 5′-CA(C/T)GA(C/T)GC(A/C)ATGCA(C/T)GG-3′ crtW101 (SEQ ID NO: 58):5′-CA(C/T)GA(C/T)GC(G/T)ATGCA(C/T)GG-3′

[0260] The C-terminal peptide H(W/H)EHH(R/L) corresponding to region IIwas used design the two 17-mer degenerate primer with the antisensesequences crtW105 (SEQ ID NO: 59) and crtW106 (SEQ ID NO: 60): crtW105(SEQ ID NO: 59): 5′-AG(G/A)TG(G/A)TG(T/C)TC(G/A)TG(G/A)TG3′ crtW106 (SEQID NO: 60): 5′-AG(G/A)TG(G/A)TG(T/C)TCCCA(G/A)TG-3′

[0261] Polymerase chain reaction: PCR was performed using the GeneAmpKit (Perkin Elmer Cetus) according to the manufacturer's instructions.The different PCR reactions contained combinations of the degenerateprimers (crtW100/crtW105 or crtW100/crtW106 or crtW100/crtW105 orcrtW101/crtW106) at a final concentration of 50 pM each, together withgenomic DNA of the bacterium E-396 (200 ng) and 2.5 units of Taqpolymerase. In total 35 cycles of PCR were performed with the followingcycle profile: 95° C. for 30 sec, 55° C. for 30 sec, 72° C. for 30 sec.PCR reactions made with the following primer combinationscrtW100/crtW105 and crtW101/crtW105 gave PCR amplification products ofapprox. 500 bp which were in accordance with the expected fragment size.The 500 bp fragment, JAPclone8, obtained in the PCR reaction usingprimers crtW101 (SEQ ID NO: 58) and crtW105 (SEQ ID NO: 59) was excisedfrom an 1.5% agarose gel and purified using the GENECLEAN Kit andsubsequently cloned into the SmaI site of pUC18 using the Sure-CloneKit, according to the manufacturer's instructions. The resulting plasmidwas named pUC18-JAPclone8 and the insert was sequenced. Comparison ofthe determined sequence to the crtW gene of Agrobacterium aurantiacum(GenBank accession n° D58420) published by Misawa et al. in 1995(WO95/18220) showed 96% identity at the nucleotide sequence level,indicating that both organisms might be closely related.

[0262] Isolation of the crt cluster of the strain E-396: Genomic DNA ofE-396 was digested overnight with different combinations of restrictionsenzymes and separated by agarose gel electrophoresis before transferringthe resulting fragments by Southern blotting onto a nitrocellulosemembrane. The blot was hybridised with a ³²P labelled 334 bp fragmentobtained by digesting the aforementioned PCR fragment JAPclone8 withBssHII and MluI. An approx. 9,4 kb EcoRI/BamHI fragment hybridizing tothe probe was identified as the most appropiate for cloning since it islong enough to potentially carry the complete crt cluster. The fragmentwas isolated and cloned into the EcoRI and BamHI sites ofpBluescriptIIKS resulting in plasmid pJAPCL544 (FIG. 29). Based on thesequence of the PCR fragment JAPclone8, two primers were synthesized toobtain more sequence information left and right hand of this fragment.FIG. 30 shows the sequence obtained containing the crtW_(E396) (fromnucleotide 40 to 768) and crtZ_(E396) (SEQ ID NO: 33) (from nucleotide765 to 1253) genes of the bacterium E-396. The nucleotide sequence ofthe crtW_(E396) (SEQ ID NO: 30) gene is shown in FIG. 31 (SEQ ID NO: 31)and the encoded amino acid sequence in FIG. 32 (SEQ ID NO: 32). Thenucleotide sequence of the crtZ_(E396) gene is shown in FIG. 33 (SEQ IDNO: 33) and the corresponding amino acid sequence in FIG. 34 (SEQ ID NO:34). Comparison to the crtW_(E396) gene of E-396 to the crtW gene of A.aurantiacum showed 97% identity at the nucleotide level and 99% identityat the amino acid level. For the crtZ gene the values were 98% and 99%,respectively.

[0263] Construction of plasmids: Both genes, crtW_(E396) andcrtZ_(E396), which are adjacent in the genome of E-396, were isolated byPCR using primer crtW107 and crtW108 and the ExpandTM High Fidelity PCRsystem of Boehringer Mannheim, according to the manufacturer'srecommendations. To facilitate the subsequent cloning steps (see sectionbelow) the primer crt107 (SEQ ID NO: 61)(5′-ATCATATGAGCGCACATGCCCTGCCCAAGGC-3′) contains an artificial NdeI site(underlined sequence) spanning the ATG start codon of the crtW_(E396)gene and the reverse primer crtW108 (SEQ ID NO: 62)(5′-ATCTCGAGTCACGTGCGCTCCTGCGCCTCGGCC-3′) has an XhoI site (underlinedsequence) just downstream of the TGA stop codon of the crtZ_(E396) gene.The final PCR reaction mix had 10 pM of each primer, 2.5 mg genomic DNAof the bacterium E-396 and 3.5 units of the TaqDNA/Pwo DNA polymerasemix. In total 35 cycles were performed with the following cycle profile:95° C., 1 min; 60° C., 1 min; 72° C. 1 min 30 sec. The PCR product ofapprox. 1250 bp was isolated from the 1% agarose gel and purified usingGENECLEAN before ligation into the SmaI site pUC18 using the Sure-CloneKit. The resulting construct was named pUC18-E396crtWZPCR (FIG. 35). Thefunctionality of both genes was tested as follows. The crtW_(E396) andcrtZ_(E396) gene were isolated from plasmid pUC18-E396crtWZPCR with NdeIand XhoI and cloned into the NdeI and SalI site of plasmidpBIIKS-crtEBIYZW resulting in plasmid pBIIKS-crtEBIY[E396WZ] (FIG. 36).E. coli TG1 cells transformed with this plasmid produced astaxanthin,adonixanthin and adonirubin but no zeaxanthin (Table 4).

[0264] Plasmid pBIIKS-crtEBIY[E396W]DZ has a truncated non-functionalcrtZ gene. FIG. 37 outlines the construction of this plasmid. The PCRreaction was run as outlined elsewhere in the text using primers crtW113(SEQ ID NO: 63)/crtW114 (SEQ ID NO: 64) and 200 ng of plasmidpUC18-JAPclone8 as template using 20 cycles with the following protocol:95° C., 45 sec/62° C., 20 sec/ 72° C., 20 sec) primer crtW113(5′-ATATACATATGGTGTCCCCCTTGGTGCGGGTGC-3′) (SEQ ID NO: 63) primer crtW114(5′-TATGGATCCGACGCGTTCCCGGACCGCCACAATGC-3′) (SEQ ID NO: 64)

[0265] The resulting 150 bp fragment was digested with BamHI and NdeIand cloned into the corresponding sites of pBIISK(+)-PCRRBScrtZresulting in the construct pBIISK(+)-PCRRBScrtZ-2. The final plasmidcarrying the genes crtE, crtB, crtI, crtY of Flavobacterium, thecrtW_(E396) gene of E-396 and a truncated non-functional crtZ gene ofFlavobacterium was obtained by isolating the MluI/NruI fragment (280 bp)of pBIISK(+)-PCRRBScrtZ-2 and cloning it, into the MluI/PmlI sites ofplasmid pBIIKS-crtEBIY[E396WZ]. E. coli cells transformed with thisplasmid produced 100% canthaxanthin (Table 4; “CRX”: cryptoxanthin;“ASX”: astaxanthin; “ADX”: adonixanthin; “ZXN”: zeaxanthin; “ECH”:echinenone; “HECH”: 3-hydroxyechinenone; “CXN”: canthaxanthin; “BCA”:β-carotene; “ADR”: adonirubin; Numbers indicate the % of the individualcarotenoid of the total carotenoids produced in the cell.). TABLE 4plasmid CRX ASX ADX ZXN ECH HECH CXN BCA ADR pBIIKScrtEBIYZW 1.1 2.044.2 52.4 <1 <1 <1 pBIIKS- 74.4 19.8 5.8 crtEBIY[E396WZ] pBIIKS- 100crtEBIY[E396W]DZ

[0266] The results of E. coli transformants carrying pBIIKScrtEBIYZW(see example 7) are also shown in Table 4 to indicate the dramaticeffect of the new genes crtW_(E396)and crtZ_(E396) on the carotenoidsproduced in these new transformants.

EXAMPLE 9

[0267] Cloning of the Remaining Crt Genes of the Gram Negative BacteriumE-396

[0268] TG1 E. coli transformants carrying the pJAPCL544 plasmid did notproduce detectable quantities of carotenoids (results not shown).Sequence analysis and comparison of the 3′ (BamHI site) of the insert ofplasmid pJAPCL544, to the crt cluster of Flavobacterium R1534 showedthat only part of the C-terminus of the crtE gene was present. Thisresult explained the lack of carotenoid production in the aforementionedtransformants. To isolate the missing N-terminal part of the gene,genomic DNA of E-396 was digested by 6 restrictions enzymes in differentcombinations: EcoRI, BamHI, PstI, SacI, SphI and XbaI and transferred bythe Southern blot technique to nitrocellulose. Hybridization of thismembrane with the ³²P radio-labelled probe (a 463 bp PstI-BamHI fragmentoriginating from the 3′ end of the insert of pJAPCL544 (FIG. 29)highlighted a ˜1300 bp-long PstI-PstI fragment. This fragment wasisolated and cloned into the PstI site of pBSIIKS(+) resulting inplasmid pBSIIKS-#1296. The sequence of the insert is shown in FIG. 38(SEQ ID NO: 35) (small cap letters refer to new sequence obtained.Capital letters show the sequence also present in the 3′ of the insertof plasmid pJAPCL544). The complete crtE gene has therefore a length of882 bp (see FIG. 39) and encodes a GGPP synthase of 294 amino acids(FIG. 40) (SEQ ID NO: 37). The crtE enzyme has 38% identity with thecrtE amino acid sequence of Erwinia herbicola and 66% withFlavobacterium R1534 WT.

[0269] Construction of plasmids: To have a plasmid carrying the completecrt cluster of E-396, the 4.7 kb MluI/BamHI fragment encoding the genescrtW, crtZ, crtY, crtI and crtB was isolated from pJAPCL544 and clonedinto the MluI/ BamHI sites of pUC18-E396crtWZPCR (see example 8). Thenew construct was named pE396CARcrtW-B (FIG. 41) and lacked theN-terminus of the crtE gene. The missing C-terminal part of the crtEgene was then introduced by ligation of the aforementioned PstI fragmentof pBIIKS-#1296 between the PstI sites of pE396CARcrtW-B. The resultingplasmid was named pE396CARcrtW-E (FIG. 41). The carotenoid distributionof the E. coli transformants carrying aforementioned plasmid were:adonixanthin (65%), astaxanthin (8%) and zeaxanthin (3%). The %indicated reflects the proportion of the total amount of carotenoidproduced in the cell.

EXAMPLE 10

[0270] Astaxanthin and adonixanthin production in Flavobacterium R1534

[0271] Among bacteria Flavobacterium may represent the best source forthe development of a fermentative production process for 3R, 3R′zeaxanthin. Derivatives of Flavobacterium sp. strain R1534, obtained byclassical mutagenesis have attracted in the past two decades wideinterest for the development of a large scale fermentative production ofzeaxanthin, although with little success. Cloning of the carotenoidbiosynthesis genes of this organism, as outlined in example 2, may allowreplacement of the classical mutagenesis approach by a more rationalone, using molecular tools to amplify the copy number of relevant genes,deregulate their expression and eliminate bottlenecks in the carotenoidbiosynthesis pathway. Furthermore, the introduction of additionalheterologous genes (e.g. crtW) will result in the production ofcarotenoids normally not synthesised by this bacterium (astaxanthin,adonirubin, adonixanthin, canthaxanthin, echinenone). The constructionof such recombinant Flavobacterium R1534 strains producing astaxanthinand adonixanthin will be outlined below.

[0272] Gene Transfer into Flavobacterium sp

[0273] Plasmid transfer by conjugative mobilization: For theconjugational crosses we constructed plasmid pRSF1010-Amp^(r), aderivative of the small (8.9 kb) broad host range plasmid RSF1010 (IncQincompatibility group) [Guerry et al., J. Bacteriol. 117:619-630 (1974)]and used E. coli S17-1 as the mobilizing strain [Priefer et al., J.Bacteriol. 163:324-330 (1985)]. In general any of the IncQ plasmids(e.g. RSF1010, R300B, R1162) may be mobilized into rifampicin resistantFlavobacterium if the transfer functions are provided by plasmids of theIncP1 group (e.g. R1, R751).

[0274] Rifampicin resistant (Rif^(r)) Flavobacterium R1534 cells wereobtained by selection on 100 mg rifampicin/ml. One resistant colony waspicked and a stock culture was made. The conjugation protocol was asfollows:

[0275] Day 1

[0276] grow 3 ml culture of Flavobacterium R1534 Rif^(r) for 24 hours at30° C. in Flavobacter medium (F-medium) (see example 1)

[0277] grow 3 ml mobilizing E. coli strain carrying the mobilizableplasmid O/N at 37° C. in LB medium. (e.g E. coli S17-1 carryingpRSF1010-Amp^(r) or E. coli TG-1 cells carrying R751 andpRSF1010-Amp^(r))

[0278] Day 2

[0279] pellet 1 ml of the Flavobacterium R1534 Rif^(r) cells andresuspend in 1ml of fresh F-medium.

[0280] pellet 1 ml of E. coli cells (see above) and resuspend in 1 ml ofLB medium.

[0281] donor and recipient cells are then mixed in a ratio of 1:1 and1:10 in an Eppendorf tube and 30 ml are then applied onto anitrocellulose filter plated on agar plates containing F-medium andincubated O/N at 30° C.

[0282] Day 3

[0283] the conjugational mixtures were washed off with F-medium andplated on F-medium containing 100 mg rifampicin and 100 mg ampicillin/mlfor selection of transconjugants and inhibition of the donor cells.

[0284] Day 6-8

[0285] Arising clones are plated once more on F-medium containing 100mgRif and 100 mg Amp/ml before analysis.

[0286] Plasmid transfer by electroporation: The protocol for theeletroporation is as follows:

[0287] 1. add 10 ml of O/N culture of Flavobacterium sp. R1534 into 500mlF-medium and incubate at 30° C. until OD600=0.8-0.1

[0288] 2. harvest cells by centrifugation at 4000 g at 4° C. for 10 min.

[0289] 3. wash cells in equal volume of ice-cold deionized water (2times)

[0290] 4. resuspend bacterial pellet in 1 ml ice-cold deionized water

[0291] 5. take 50 ml of cells for electroporation with 0.1 mg of plasmidDNA

[0292] 6. electroporation was done using field strengths between 15 and25 kV/cm and 1-3 ms.

[0293] 7. after electroporation cells were immediately diluted in 1 mlof F-medium and incubated for 2 hours at 30° C. at 180 rpm beforeplating on F-medium plates containing the respective selectiveantibioticum.

[0294] Plasmid constructions: Plasmid pRSF101-Amp^(r) was obtained bycloning the Amp^(r) gene of pBR322 between the EcoRI/NotI sites ofRSF1010. The Amp^(r) gene originates from pBR322 and was isolated by PCRusing primers AmpR1 (SEQ ID NO: 65) and AmpR2 (SEQ ID NO: 66) as shownin FIG. 42.

[0295] AmpR1 (SEQ ID NO: 65)

[0296] 5′-TATATCGGCCGACTAGTAAGCTTCAAAAAGGATCTTCACCTAG-3′ the underlinedsequence contains the introduced restriction sites for EagI, SpeI andHindIII to facilitate subsequent constructions.

[0297] AmpR2 (SEQ ID NO: 66)

[0298] 5′-ATATGAATTCAATAATATTGAAAAAGGAAG-3′ the underlined sequencecorresponds to an introduced EcoRI restriction site to facilitatecloning into RSF1010 (see FIG. 42).

[0299] The PCR reaction mix had 10 pM of each primer (AmpR1 (SEQ ID NO:65)/ AmpR2 (SEQ ID NO: 66)), 0.5 mg plasmid pBR322 and 3.5 units of theTaqDNA/Pwo DNA polymerase mix. In total 35 amplification cycles weremade with the profile: 95° C., 45 sec; 59° C., 45 sec, 72° C., 1 min.The PCR product of approx. 950 was extracted once with phenol/chloroformand precipitated with 0.3 M NaAcetate and 2 vol. Ethanol. The pellet wasresuspended in H₂O and digested with EcoRI and EagI O/N. The digestionwas separated by electrophoresis and the fragment isolated from the 1%agarose gel and purified using GENECLEAN before ligation into the EcoRIand NotI sites of RSF1010. The resulting plasmid was namedpRSF1010-Amp^(r)(FIG. 42).

[0300] Plasmid RSF1010-Amprcrt1 was obtained by isolating theHindIII/NotI fragment of pBIIKS-crtEBIY[E396WZ] and cloning it betweenthe HindIII/EagI sites of RSF1010-Amp^(r) (FIG. 43). The resultingplasmid RSF1010-Ampr-crt1 carries crtW_(E396), crtZ_(E396), crtY genesand the N-terminus of the crtI gene (non-functional). PlasmidRSF1010-Ampr-crt2 carrying a complete crt cluster composed of the genescrtW_(E396) and crtZ_(E396) of E-396 and the crtY, crtI, crtB and crtEof Flavobacterium R1534 was obtained by isolating the large HindIII/XbaIfragment of pBIIKS-crtEBIY[E396WZ] and cloning it into the SpeI/HindIIIsites of RSF1010-Amp^(r) (FIG. 43).

[0301] Flavobacterium R1534 transformants carrying either plasmidRSF1010-Amp^(r), Plasmid RSF1010-Amp^(r)-crt1 or PlasmidRSF1010-Amp^(r)-crt2 were obtained by conjugation as outlined aboveusing E. coli S17-1 as mobilizing strain.

[0302] Comparison of the carotenoid production of two flavobacteriumtransformants: Overnight cultures of the individual transformants werediluted into 20 ml fresh F-medium to have a final starting OD600 of 0.4.Cells were harvested after growing for 48 hours at 30° C. and carotenoidcontents were analysed as outlined in example 7. Table 5 shows theresult of the three control cultures Flavobacterium [R1534 WT], [R1534WT RifR] (rifampicin resistant) and [R1534WT Rifr RSF1010-AmpR] (carriesthe RSF1010-Amp^(r) plasmid) and the two transformants [R1534 WTRSF1010-AmpR-crt1] and [R1534 WT RSF1010-AmpR-crt2]. Both lattertransformants are able to synthesise astaxanthin and adonixanthin butlittle zeaxanthin. Most interesting is the [R1534 WT RSF1010-AmpR-crt2]Flavobacterium transformant which produces approx. 4 times morecarotenoids than the R1534 WT. This increase in total carotenoidproduction is most likely due to the increase of the number ofcarotenoid biosynthesis clusters present in these cell (e.g. correspondsto the total copy number of plasmids in the cell). TABLE 5 totalcarotenoid carotenoids % of total content in % Transformant dry weightof dry weight R1534 WT 0.039% b-Carotin 0.001% 0.06% b-Cryptoxanthin0.018% Zeaxanthin R1534 Rifr 0.036% b-Carotin 0.002% 0.06%b-Cryptoxanthin 0.022% Zeaxanthin R1534 Rifr 0.021% b-Carotin 0.002%0.065% [RSF1010-Ampr] b-Cryptoxanthin 0.032% Zeaxanthin R1534 Rifr0.022% Astaxanthin 0.075% 0.1% [RSF1010-Ampr-crt1] Adonixanthin 0.004%Zeaxanthin R1534 Rifr 0.132% b-Carotin 0.006% 0.235% [RSF1010-Ampr-crt2]Echinenon 0.004% Hydroxyechinenon 0.003% b-Cryptoxanthin 0.044%Astaxanthin 0.039% Adonixanthin 0.007% Zeaxanthin

[0303]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 66 <210> SEQ ID NO 1<211> LENGTH: 8625 <212> TYPE: DNA <213> ORGANISM: Flavobacterium sp.R1534 <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(8348)..(8349) <221> NAME/KEY: unsure <222> LOCATION: (8539)..(8540)<221> NAME/KEY: unsure <222> LOCATION: (8581) <221> NAME/KEY: unsure<222> LOCATION: (8590) <221> NAME/KEY: unsure <222> LOCATION: (8592)<221> NAME/KEY: unsure <222> LOCATION: (8602)..(8604) <400> SEQUENCE: 1ggatccgcgc ctggccgttc gcgatcagca gccgcccttg cggatcggtc agcatcatcc 60ccatgaaccg cagcgcacga cgcagcgcgc gccccagatc gggcgcgtcc agcacggcat 120gcgccatcat cgcgaaggcc cccggcggca tggggcgcgt gcccattccg aagaactcgc 180agcctgtccg ctgcgcaagg tcgcgccaga tcgcgccgta ttccgatgca gtgacgggcc 240cgatgcgcgt gggcccgccc tgccccgccg ccaccagcgc atcgcgcacg aacccttccg 300agatgatgtg ctgatccatg gcccgtcatt gcaaaaccga tcaccgatcc tgtcgcgtga 360tggcattgtt tgcaatgccc cgagggctag gatggcgcga aggatcaagg gggggagaga 420catggaaatc gagggacggg tctttgtcgt cacgggcgcc gcatcgggtc tgggggcggc 480ctcggcgcgg atgctggccc aaggcggcgc gaaggtcgtg ctggccgatc tggcggaacc 540gaaggacgcg cccgaaggcg cggttcacgc ggcctgcgac gtgaccgacg cgaccgctgc 600gcagacggcc atcgcgctgg cgaccgaccg cttcggcagg ctggacggcc ttgtgaactg 660cgcgggcatc gcgccggccg aacggatgct gggccgcgac gggccgcatg gactggacag 720ctttgcccgt gcggtcacga tcaacctgat cggcagcttc aacatggccc gccttgcagc 780cgaggcgatg gcccggaacg agcccgtccg gggcgagcgt ggcgtgatcg tcaacacggc 840ctcgatcgcg gcgcaggacg gacagatcgg acaggtcgcc tatgcggcca gcaaggcggg 900cgtggcgggc atgacgctgc cgatggcccg cgaccttgcg cggcacggca tccgcgtcat 960gaccatcgcg cccggcatct tccgcacccc gatgctggag gggctgccgc aggacgttca 1020ggacagcctg ggcgcggcgg tgcccttccc ctcgcggctg ggagagccgt cggaatacgc 1080ggcgctgttg caccacatca tcgcgaaccc catgctgaac ggagaggtca tccgcctcga 1140cggcgcattg cgcatggccc ccaagtgaag gagcgtttca tggaccccat cgtcatcacc 1200ggcgcgatgc gcaccccgat gggggcattc cagggcgatc ttgccgcgat ggatgccccg 1260acccttggcg cggacgcgat ccgcgccgcg ctgaacggcc tgtcgcccga catggtggac 1320gaggtgctga tgggctgcgt cctcgccgcg ggccagggtc aggcaccggc acgtcaggcg 1380gcgcttggcg ccggactgcc gctgtcgacg ggcacgacca ccatcaacga gatgtgcgga 1440tcgggcatga aggccgcgat gctgggccat gacctgatcg ccgcgggatc ggcgggcatc 1500gtcgtcgccg gcgggatgga gagcatgtcg aacgccccct acctgctgcc caaggcgcgg 1560tcggggatgc gcatgggcca tgaccgtgtg ctggatcaca tgttcctcga cgggttggag 1620gacgcctatg acaagggccg cctgatgggc accttcgccg aggattgcgc cggcgatcac 1680ggtttcaccc gcgaggcgca ggacgactat gcgctgacca gcctggcccg cgcgcaggac 1740gccatcgcca gcggtgcctt cgccgccgag atcgcgcccg tgaccgtcac ggcacgcaag 1800gtgcagacca ccgtcgatac cgacgagatg cccggcaagg cccgccccga gaagatcccc 1860catctgaagc ccgccttccg tgacggtggc acggtcacgg cggcgaacag ctcgtcgatc 1920tcggacgggg cggcggcgct ggtgatgatg cgccagtcgc aggccgagaa gctgggcctg 1980acgccgatcg cgcggatcat cggtcatgcg acccatgccg accgtcccgg cctgttcccg 2040acggccccca tcggcgcgat gcgcaagctg ctggaccgca cggacacccg ccttggcgat 2100tacgacctgt tcgaggtgaa cgaggcattc gccgtcgtcg ccatgatcgc gatgaaggag 2160cttggcctgc cacacgatgc cacgaacatc aacggcgggg cctgcgcgct tgggcatccc 2220atcggcgcgt cgggggcgcg gatcatggtc acgctgctga acgcgatggc ggcgcggggc 2280gcgacgcgcg gggccgcatc cgtctgcatc ggcgggggcg aggcgacggc catcgcgctg 2340gaacggctga gctaattcat ttgcgcgaat ccgcgttttt cgtgcacgat gggggaaccg 2400gaaacggcca cgcctgttgt ggttgcgtcg acctgtcttc gggccatgcc cgtgacgcga 2460tgtggcaggc gcatggggcg ttgccgatcc ggtcgcatga ctgacgcaac gaaggcaccg 2520atgacgccca agcagcaatt ccccctacgc gatctggtcg agatcaggct ggcgcagatc 2580tcgggccagt tcggcgtggt ctcggccccg ctcggcgcgg ccatgagcga tgccgccctg 2640tcccccggca aacgctttcg cgccgtgctg atgctgatgg tcgccgaaag ctcgggcggg 2700gtctgcgatg cgatggtcga tgccgcctgc gcggtcgaga tggtccatgc cgcatcgctg 2760atcttcgacg acatgccctg catggacgat gccaggaccc gtcgcggtca gcccgccacc 2820catgtcgccc atggcgaggg gcgcgcggtg cttgcgggca tcgccctgat caccgaggcc 2880atgcggattt tgggcgaggc gcgcggcgcg acgccggatc agcgcgcaag gctggtcgca 2940tccatgtcgc gcgcgatggg accggtgggg ctgtgcgcag ggcaggatct ggacctgcac 3000gcccccaagg acgccgccgg gatcgaacgt gaacaggacc tcaagaccgg cgtgctgttc 3060gtcgcgggcc tcgagatgct gtccattatt aagggtctgg acaaggccga gaccgagcag 3120ctcatggcct tcgggcgtca gcttggtcgg gtcttccagt cctatgacga cctgctggac 3180gtgatcggcg acaaggccag caccggcaag gatacggcgc gcgacaccgc cgcccccggc 3240ccaaagggcg gcctgatggc ggtcggacag atgggcgacg tggcgcagca ttaccgcgcc 3300agccgcgcgc aactggacga gctgatgcgc acccggctgt tccgcggggg gcagatcgcg 3360gacctgctgg cccgcgtgct gccgcatgac atccgccgca gcgcctaggc gcgcggtcgg 3420gtccacaggc cgtcgcggct gatttcgccg ccgcgcaggc gcgatgcggc cgcgtccaag 3480cctccgcgcg ccagaagccc gatcttggca gccttcgacg tgctgatccg ctggcgatag 3540gcctcggggc caccctgccg gatgcgcgtc ccgattgcgc gatagatacg cagcgcggcg 3600gcgatcgacc acgcgcagcg cggcggcaga tgcggaagcc cctgccgcgc cgaggcataa 3660tagggctcgg ccgcgtcaag caggcggatg atgacggaat agagcgcgtc cgaaggcacc 3720ggaccctcaa ccgtcgcccc cgcctcggcc agccagtcgg caggcagata gcagcgcccg 3780atggcggcat cgtcgatcac gtcgcgagcg atgttcgtca gctggaacgc aaggcccaga 3840tcgcaggcgc gatccagcac cgcatcgtcc tgcacgccca tcacccgcgc catcatcacg 3900cccacgaccc ccgcgacgtg gtaggaatat tccagcacgt catccaggct gcggtattcg 3960cgatccgcga catccatcgc gaaaccctcg atcaggtcca tcggccaaag gtccgggaaa 4020tcatgccgcc gggcgacctg gcgcagcgcc gcgaagggcg gcgacatcgg gccgtcctcg 4080tgcagcgcgg ccagcgtgtc ggcgcgcagc gcccccagcc gcgcctgtgg gtcgccgccc 4140gcctcggggg cagaacccat cacctgcccg tcgatcacgt catccgcatg cctgcaccag 4200gcatagagca tgaccgtatc ctcgcggatg ccgggcggca tcagcttggc cgcctgcgcg 4260aagctttgcg aaccctgcgc gatggccgct tcggaagtcg ccgtcagatc ggtcatgcga 4320cggccaggtc cgacagcatg acctgcgccg tggccttggc gctgccaacg acacccggga 4380tgcccgcacc cggatgcgtg cccgccccca cgatgtagaa gttcgggatc gcgcggtcgc 4440ggttatgcgg gcggaaccag gcggattgcg tcaggatcgg ctcgaccgag aaggcgctgc 4500cgtgatgggc cgacagttcg gtgctgaaat cggcggggct gaagatgcgg ctgacggtca 4560ggtgcttgcg caggtcgggg atggcgcggc gctccagttc ctcgaagatg cgctcggcat 4620agcccggggc ctcggcttcc caatcgacat cggcgcggcc cagatgcgga acgggcgcaa 4680ggacgtaatg cgtggacatc ccctcggggg ccaggctggg atcggtcacg cagggcgaat 4740gcagatacat cgagaaatcg tccggcaggc gtggcccgtt gaagatctcg ttcaccagcc 4800ccttgtagcg cgggccgaag atgacgctgt ggtgggccag gttctcgggg cgcttggaca 4860ggccgaaatg cagcacgaac agcgacatcg accagcgctg ccggttcagg atcgcggcct 4920tggtgcgccc gcggcgggta tggcccagca ggtcgcgata gctgtgcatc acgtcgccgt 4980tgctggccac cgtatccgcg cgcaactgcc gcccgtccag cagcgtgacg cccgtggcgc 5040gatcgccctc ggtgtcgatc cgcgtgacgc gggcattcag cagcagcgtg ccgccaagac 5100gctcgaacag ggcgaccatg cccgcgacca gctggttggt gccgcccttg gcgaaccaga 5160cgccgccgcg ccgttccagc gcatggatca gcgcatagat cgagctggtc gaaaacgggt 5220tcccgccgac cagcagcgtg tggaacgaga aggcctgccg cagatgcggg tcctggatga 5280agcgcgccac catgctgtgg accgagcggt atgcctgcag gcgcatcagc gccggcgcgg 5340cgttcagcat ctggcccagc ttcaggaagg gcgtggtccc cagcttcaga tacccctcgc 5400gatagacctc ctcggcgtaa tcgtggaagc ggcgatagcc atcgacatcg gcgggattga 5460aggaggcgac ctggcggatc agctcgtcgt cgtcgttcac gtattcgaag ctgcggccgt 5520ccgcccatgt cagccggtag aagggcgaga ccggcagcag cgtcacgtca cgctccatcg 5580gttggccgct gagggcccac agctctcgca ggctgtcggg gtcggtcacg accgtcgggc 5640ctgcatcgaa gacgtggccc tgatcgttcc agacataggc gcggccgccg ggcttgtcgc 5700gggcctcgac gatggtggtc gcgatgccgg ccgattgcag gcggatggca agcgcaagcc 5760cgccgaaacc tgcgccgatg acgatggcgg aactcatgct ctctcctgca gcagggggcg 5820ttcgggcagg cagcgcacgg cctgcgacag cggaatgggc gggcgtccgg tgacgatgcg 5880aagccggtcg gccaatgtca ggcgcccggc atagaagcgc tcgatcagcg gctgcggcag 5940gcggtagaac cgctgcagca ggcgatagcg acggtcgggc gggcagccgc ggaacagcat 6000ccggttcagc agccgcagga agcggtcgcg atccgcgcga tcgatggccc agccgcgcac 6060cgcgcgacgg gcggacgcgg tcgtcaggtc gcgcgccgcg atggcatccg cgacctgcgc 6120ggcatagggc agcgaatatc cggtgacggg gtggaacagc cctgccccca gcccaaccgg 6180caccgccccc tgcgcgtggt cgcgccagaa gcctatggcg tcatgggcca gcgcgatggg 6240caggatgccc ctttcgcgcc gcatctcctg cccggtccag ccccgcctgg cggcatagtc 6300cagcgacgcc tgcgccagcg cgccatcgtc cagatcgccg ccgtcgctgt agcgcgtatc 6360ctcgatcagg atgcgggtgg gactgaaggg cagcagatag atgaagcggt acccgtccat 6420ctgcggaacg gtcgcgtcca tgatcatcgg gcgctcgacg ccatgggggg cgtcggtctc 6480gatctcgacg cccacgaatt tctggaaacc cacggtcagg tgcggggtct cgacggcacc 6540acgggcgtcg atcacgcagg cagcctcgat ccgcgagccg tccgtcagcg tcgcgccggt 6600atcgtccagc gtcgcgacat gcgtattcca ccgcagatcg acaccctgca gcagcccgat 6660cagcgcgccc gcctcgatcg agccatagcc tgtcgtcagg cggcgcgaat ggtcgggaaa 6720cgcgacctcc tgatccgtcc attcgccgcg acgaatgggc gacaggcgcg ccagccattc 6780gggcgaaaga tccgtgtcgt ggcaggacca ggtgtgctgg tccgaggggc cggaccgcgc 6840gtcgagcatc acgatgcgcg catccggtct gcggtcgcga acggcaagcg cgatcagcgc 6900accggacagc cccgcgcccg cgatcagcag atcatggctc atgtattgcg atccgcccct 6960tcgcggtcct tcagcagcgc gcccgagcgt ttcagctctg ccttgaggct gtcgaccgag 7020ggcgcccaga tgaaaccgaa gctgacgcag ttctcgcggc catggaccgc gtgatgcatc 7080ctgtgtgcct ggtagacgcg acgaagatag ccgcgcttgg ggacatagcg gaacggccag 7140cgcccatgca ccaagccgtc atgcaggaaa tagtagatca gcccgtagca ggtgaccccc 7200accgccagcc accaggccag atccgacccc atcgcgccga tcgcgaacag cacgatcgag 7260attaccgcga agatgacgcc atagaggtcg ttcttctcga gcgcgtggtc gtgatcctcg 7320tcgtggtgcg atttatgcca gccccagccc agggggccat gcatgatcca ccgatggacg 7380gagtaggccg tcagctccat cgcggcgacg gtcaggatga cggtcaggat tgcggcccaa 7440gtgctcatgc cggccccttg cttgatatga cagggaacag gctacgctgc cgcgcggtgc 7500atgaccagcc catcggggtg cgaccaaagg gcatcgcgtg acatctgcgt tcagggctca 7560taggcggatc atccgtgaca ttcgccgccg aacgcggcag gcgcatcacg cgttccgtcg 7620ctggaaatat taatgttttc ccgaagatgg tcggggcgag aggattcgaa cctccgacct 7680acggtaccca aaaccgtcgc gctaccaggc tgcgctacgc cccgactgcg gaaggcttta 7740gccgattgtt ccggcaaggg aaagacctag tcgcaggcca ggaccgcatt gtcgcccatg 7800cccggatgcg ccatcggctg accgggcttc aggccaaggc gatccgcctc tccgcccgcg 7860atttcgagga cgaacagccg gtcggggtcc ggatcgccga ccgccgcgcc cggaatgggc 7920gtctcgtcca gcgggcgcgc attgcggtgg atgtggcgga tgacgccggt ttcatccgca 7980aagaccatgt ccagcgggat cagtgtgttg cgcatccaga aggacaccgg ctggggcgat 8040tcgtagatga acagcattcc ggtgcccgca ggcagctcct tgcggaacat caggccctgc 8100gcgcgctctt cggggctgtc cgcgacctcg acccgaaacc cgagcgtttc cgcaccggta 8160tcgacgacaa gactgccggg cgcgcattcc accgccgccg cggcggcggg catcaggacc 8220gcaagaagcg ctgcggcctt actcggccac atgggcaaga taggactgct cggcgccgag 8280atcctgctga ccctgcgcat cctcgttccg gtcatgcagc gccaggtccc atgccgcgat 8340ctgcgcgnnc atcagcccgc gcggaccctc gacgacgcgg aggcagatcg cctcgccgat 8400cacgaggtcc gagaagccgg aatgacggag cacctcgata tggatgaaca cgtcctcggg 8460gtggccgaag atgttggcga accgggaaaa ggcccttggc cttgtcgaac cacttgacgc 8520gggccggacg cagcggcann cgtccagatg ctcgatcacc tcggcatcca gatcggcgat 8580nggggggtgn cngtcgcttt cnnncggttc gatcgacagg acctc 8625 <210> SEQ ID NO 2<211> LENGTH: 295 <212> TYPE: PRT <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 2 Met Thr Pro Lys Gln Gln Phe Pro Leu Arg Asp LeuVal Glu Ile Arg 1 5 10 15 Leu Ala Gln Ile Ser Gly Gln Phe Gly Val ValSer Ala Pro Leu Gly 20 25 30 Ala Ala Met Ser Asp Ala Ala Leu Ser Pro GlyLys Arg Phe Arg Ala 35 40 45 Val Leu Met Leu Met Val Ala Glu Ser Ser GlyGly Val Cys Asp Ala 50 55 60 Met Val Asp Ala Ala Cys Ala Val Glu Met ValHis Ala Ala Ser Leu 65 70 75 80 Ile Phe Asp Asp Met Pro Cys Met Asp AspAla Arg Thr Arg Arg Gly 85 90 95 Gln Pro Ala Thr His Val Ala His Gly GluGly Arg Ala Val Leu Ala 100 105 110 Gly Ile Ala Leu Ile Thr Glu Ala MetArg Ile Leu Gly Glu Ala Arg 115 120 125 Gly Ala Thr Pro Asp Gln Arg AlaArg Leu Val Ala Ser Met Ser Arg 130 135 140 Ala Met Gly Pro Val Gly LeuCys Ala Gly Gln Asp Leu Asp Leu His 145 150 155 160 Ala Pro Lys Asp AlaAla Gly Ile Glu Arg Glu Gln Asp Leu Lys Thr 165 170 175 Gly Val Leu PheVal Ala Gly Leu Glu Met Leu Ser Ile Ile Lys Gly 180 185 190 Leu Asp LysAla Glu Thr Glu Gln Leu Met Ala Phe Gly Arg Gln Leu 195 200 205 Gly ArgVal Phe Gln Ser Tyr Asp Asp Leu Leu Asp Val Ile Gly Asp 210 215 220 LysAla Ser Thr Gly Lys Asp Thr Ala Arg Asp Thr Ala Ala Pro Gly 225 230 235240 Pro Lys Gly Gly Leu Met Ala Val Gly Gln Met Gly Asp Val Ala Gln 245250 255 His Tyr Arg Ala Ser Arg Ala Gln Leu Asp Glu Leu Met Arg Thr Arg260 265 270 Leu Phe Arg Gly Gly Gln Ile Ala Asp Leu Leu Ala Arg Val LeuPro 275 280 285 His Asp Ile Arg Arg Ser Ala 290 295 <210> SEQ ID NO 3<211> LENGTH: 303 <212> TYPE: PRT <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 3 Met Thr Asp Leu Thr Ala Thr Ser Glu Ala Ala IleAla Gln Gly Ser 1 5 10 15 Gln Ser Phe Ala Gln Ala Ala Lys Leu Met ProPro Gly Ile Arg Glu 20 25 30 Asp Thr Val Met Leu Tyr Ala Trp Cys Arg HisAla Asp Asp Val Ile 35 40 45 Asp Gly Gln Val Met Gly Ser Ala Pro Glu AlaGly Gly Asp Pro Gln 50 55 60 Ala Arg Leu Gly Ala Leu Arg Ala Asp Thr LeuAla Ala Leu His Glu 65 70 75 80 Asp Gly Pro Met Ser Pro Pro Phe Ala AlaLeu Arg Gln Val Ala Arg 85 90 95 Arg His Asp Phe Pro Asp Leu Trp Pro MetAsp Leu Ile Glu Gly Phe 100 105 110 Ala Met Asp Val Ala Asp Arg Glu TyrArg Ser Leu Asp Asp Val Leu 115 120 125 Glu Tyr Ser Tyr His Val Ala GlyVal Val Gly Val Met Met Ala Arg 130 135 140 Val Met Gly Val Gln Asp AspAla Val Leu Asp Arg Ala Cys Asp Leu 145 150 155 160 Gly Leu Ala Phe GlnLeu Thr Asn Ile Ala Arg Asp Val Ile Asp Asp 165 170 175 Ala Ala Ile GlyArg Cys Tyr Leu Pro Ala Asp Trp Leu Ala Glu Ala 180 185 190 Gly Ala ThrVal Glu Gly Pro Val Pro Ser Asp Ala Leu Tyr Ser Val 195 200 205 Ile IleArg Leu Leu Asp Ala Ala Glu Pro Tyr Tyr Ala Ser Ala Arg 210 215 220 GlnGly Leu Pro His Leu Pro Pro Arg Cys Ala Trp Ser Ile Ala Ala 225 230 235240 Ala Leu Arg Ile Tyr Arg Ala Ile Gly Thr Arg Ile Arg Gln Gly Gly 245250 255 Pro Glu Ala Tyr Arg Gln Arg Ile Ser Thr Ser Lys Ala Ala Lys Ile260 265 270 Gly Leu Leu Ala Arg Gly Gly Leu Asp Ala Ala Ala Ser Arg LeuArg 275 280 285 Gly Gly Glu Ile Ser Arg Asp Gly Leu Trp Thr Arg Pro ArgAla 290 295 300 <210> SEQ ID NO 4 <211> LENGTH: 494 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 4 Met Ser SerAla Ile Val Ile Gly Ala Gly Phe Gly Gly Leu Ala Leu 1 5 10 15 Ala IleArg Leu Gln Ser Ala Gly Ile Ala Thr Thr Ile Val Glu Ala 20 25 30 Arg AspLys Pro Gly Gly Arg Ala Tyr Val Trp Asn Asp Gln Gly His 35 40 45 Val PheAsp Ala Gly Pro Thr Val Val Thr Asp Pro Asp Ser Leu Arg 50 55 60 Glu LeuTrp Ala Leu Ser Gly Gln Pro Met Glu Arg Asp Val Thr Leu 65 70 75 80 LeuPro Val Ser Pro Phe Tyr Arg Leu Thr Trp Ala Asp Gly Arg Ser 85 90 95 PheGlu Tyr Val Asn Asp Asp Asp Glu Leu Ile Arg Gln Val Ala Ser 100 105 110Phe Asn Pro Ala Asp Val Asp Gly Tyr Arg Arg Phe His Asp Tyr Ala 115 120125 Glu Glu Val Tyr Arg Glu Gly Tyr Leu Lys Leu Gly Thr Thr Pro Phe 130135 140 Leu Lys Leu Gly Gln Met Leu Asn Ala Ala Pro Ala Leu Met Arg Leu145 150 155 160 Gln Ala Tyr Arg Ser Val His Ser Met Val Ala Arg Phe IleGln Asp 165 170 175 Pro His Leu Arg Gln Ala Phe Ser Phe His Thr Leu LeuVal Gly Gly 180 185 190 Asn Pro Phe Ser Thr Ser Ser Ile Tyr Ala Leu IleHis Ala Leu Glu 195 200 205 Arg Arg Gly Gly Val Trp Phe Ala Lys Gly GlyThr Asn Gln Leu Val 210 215 220 Ala Gly Met Val Ala Leu Phe Glu Arg LeuGly Gly Thr Leu Leu Leu 225 230 235 240 Asn Ala Arg Val Thr Arg Ile AspThr Glu Gly Asp Arg Ala Thr Gly 245 250 255 Val Thr Leu Leu Asp Gly ArgGln Leu Arg Ala Asp Thr Val Ala Ser 260 265 270 Asn Gly Asp Val Met HisSer Tyr Arg Asp Leu Leu Gly His Thr Arg 275 280 285 Arg Gly Arg Thr LysAla Ala Ile Leu Asn Arg Gln Arg Trp Ser Met 290 295 300 Ser Leu Phe ValLeu His Phe Gly Leu Ser Lys Arg Pro Glu Asn Leu 305 310 315 320 Ala HisHis Ser Val Ile Phe Gly Pro Arg Tyr Lys Gly Leu Val Asn 325 330 335 GluIle Phe Asn Gly Pro Arg Leu Pro Asp Asp Phe Ser Met Tyr Leu 340 345 350His Ser Pro Cys Val Thr Asp Pro Ser Leu Ala Pro Glu Gly Met Ser 355 360365 Thr His Tyr Val Leu Ala Pro Val Pro His Leu Gly Arg Ala Asp Val 370375 380 Asp Trp Glu Ala Glu Ala Pro Gly Tyr Ala Glu Arg Ile Phe Glu Glu385 390 395 400 Leu Glu Arg Arg Ala Ile Pro Asp Leu Arg Lys His Leu ThrVal Ser 405 410 415 Arg Ile Phe Ser Pro Ala Asp Phe Ser Thr Glu Leu SerAla His His 420 425 430 Gly Ser Ala Phe Ser Val Glu Pro Ile Leu Thr GlnSer Ala Trp Phe 435 440 445 Arg Pro His Asn Arg Asp Arg Ala Ile Pro AsnPhe Tyr Ile Val Gly 450 455 460 Ala Gly Thr His Pro Gly Ala Gly Ile ProGly Val Val Gly Ser Ala 465 470 475 480 Lys Ala Thr Ala Gln Val Met LeuSer Asp Leu Ala Val Ala 485 490 <210> SEQ ID NO 5 <211> LENGTH: 382<212> TYPE: PRT <213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE:5 Met Ser His Asp Leu Leu Ile Ala Gly Ala Gly Leu Ser Gly Ala Leu 1 5 1015 Ile Ala Leu Ala Val Arg Asp Arg Arg Pro Asp Ala Arg Ile Val Met 20 2530 Leu Asp Ala Arg Ser Gly Pro Ser Asp Gln His Thr Trp Ser Cys His 35 4045 Asp Thr Asp Leu Ser Pro Glu Trp Leu Ala Arg Leu Ser Pro Ile Arg 50 5560 Arg Gly Glu Trp Thr Asp Gln Glu Val Ala Phe Pro Asp His Ser Arg 65 7075 80 Arg Leu Thr Thr Gly Tyr Gly Ser Ile Glu Ala Gly Ala Leu Ile Gly 8590 95 Leu Leu Gln Gly Val Asp Leu Arg Trp Asn Thr His Val Ala Thr Leu100 105 110 Asp Asp Thr Gly Ala Thr Leu Thr Asp Gly Ser Arg Ile Glu AlaAla 115 120 125 Cys Val Ile Asp Ala Arg Gly Ala Val Glu Thr Pro His LeuThr Val 130 135 140 Gly Phe Gln Lys Phe Val Gly Val Glu Ile Glu Thr AspAla Pro His 145 150 155 160 Gly Val Glu Arg Pro Met Ile Met Asp Ala ThrVal Pro Gln Met Asp 165 170 175 Gly Tyr Arg Phe Ile Tyr Leu Leu Pro PheSer Pro Thr Arg Ile Leu 180 185 190 Ile Glu Asp Thr Arg Tyr Ser Asp GlyGly Asp Leu Asp Asp Gly Ala 195 200 205 Leu Ala Gln Ala Ser Leu Asp TyrAla Ala Arg Arg Gly Trp Thr Gly 210 215 220 Gln Glu Met Arg Arg Glu ArgGly Ile Leu Pro Ile Ala Leu Ala His 225 230 235 240 Asp Ala Ile Gly PheTrp Arg Asp His Ala Gln Gly Ala Val Pro Val 245 250 255 Gly Leu Gly AlaGly Leu Phe His Pro Val Thr Gly Tyr Ser Leu Pro 260 265 270 Tyr Ala AlaGln Val Ala Asp Ala Ile Ala Ala Arg Asp Leu Thr Thr 275 280 285 Ala SerAla Arg Arg Ala Val Arg Gly Trp Ala Ile Asp Arg Ala Asp 290 295 300 ArgAsp Arg Phe Leu Arg Leu Leu Asn Arg Met Leu Phe Arg Gly Cys 305 310 315320 Pro Pro Asp Arg Arg Tyr Arg Leu Leu Gln Arg Phe Tyr Arg Leu Pro 325330 335 Gln Pro Leu Ile Glu Arg Phe Tyr Ala Gly Arg Leu Thr Leu Ala Asp340 345 350 Arg Leu Arg Ile Val Thr Gly Arg Pro Pro Ile Pro Leu Ser GlnAla 355 360 365 Val Arg Cys Leu Pro Glu Arg Pro Leu Leu Gln Glu Arg Ala370 375 380 <210> SEQ ID NO 6 <211> LENGTH: 169 <212> TYPE: PRT <213>ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 6 Met Ser Thr Trp AlaAla Ile Leu Thr Val Ile Leu Thr Val Ala Ala 1 5 10 15 Met Glu Leu ThrAla Tyr Ser Val His Arg Trp Ile Met His Gly Pro 20 25 30 Leu Gly Trp GlyTrp His Lys Ser His His Asp Glu Asp His Asp His 35 40 45 Ala Leu Glu LysAsn Asp Leu Tyr Gly Val Ile Phe Ala Val Ile Ser 50 55 60 Ile Val Leu PheAla Ile Gly Ala Met Gly Ser Asp Leu Ala Trp Trp 65 70 75 80 Leu Ala ValGly Val Thr Cys Tyr Gly Leu Ile Tyr Tyr Phe Leu His 85 90 95 Asp Gly LeuVal His Gly Arg Trp Pro Phe Arg Tyr Val Pro Lys Arg 100 105 110 Gly TyrLeu Arg Arg Val Tyr Gln Ala His Arg Met His His Ala Val 115 120 125 HisGly Arg Glu Asn Cys Val Ser Phe Gly Phe Ile Trp Ala Pro Ser 130 135 140Val Asp Ser Leu Lys Ala Glu Leu Lys Arg Ser Gly Ala Leu Leu Lys 145 150155 160 Asp Arg Glu Gly Ala Asp Arg Asn Thr 165 <210> SEQ ID NO 7 <211>LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer #100 <400> SEQUENCE: 7 tatatactag taagaggaga aattacatatgacgcccaag cagcagcaat tc 52 <210> SEQ ID NO 8 <211> LENGTH: 32 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer #101 <400>SEQUENCE: 8 tatatacccg ggtcagccgc gacggcctgt gg 32 <210> SEQ ID NO 9<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer #104 <400> SEQUENCE: 9 tatatgaatt caagaggaga aattacatatgagcacttgg gccgcaatcc 50 <210> SEQ ID NO 10 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer #105 <400>SEQUENCE: 10 gtttcagctc tgccttgagg c 21 <210> SEQ ID NO 11 <211> LENGTH:62 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Primer MUT1<400> SEQUENCE: 11 gcgaaggggc ggatcgcaat acgtgaaagg aggacacgtgatgagccatg atctgctgat 60 cg 62 <210> SEQ ID NO 12 <211> LENGTH: 63 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer MUT2 <400>SEQUENCE: 12 gccccctgct gcaggagaga gcttgaaagg aggcaattga gatgagttccgccatcgtca 60 tcg 63 <210> SEQ ID NO 13 <211> LENGTH: 70 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer MUT3 <400>SEQUENCE: 13 ggtcatgctg tcggacctgg ccgtcgcttg aaaggaggat ccaatcatgaccgatctgac 60 ggcgacttcc 70 <210> SEQ ID NO 14 <211> LENGTH: 44 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer MUT5 <400>SEQUENCE: 14 atatatctca attgcctcct ttcaagctct ctcctgcagc aggg 44 <210>SEQ ID NO 15 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer MUT6 <400> SEQUENCE: 15 atgattggatcctcctttca agcgacggcc aggtccgaca gc 42 <210> SEQ ID NO 16 <211> LENGTH:22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: PrimerCAR17 <400> SEQUENCE: 16 cagaacccat cacctgcccg tc 22 <210> SEQ ID NO 17<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer CATe <400> SEQUENCE: 17 cgcgaattct cgccggcaat agttacc27 <210> SEQ ID NO 18 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer CAT4 <400> SEQUENCE: 18 gtcacatgcatgcatgttac gagctcataa gcatgtgacg tcttcaacta acggggcagg 60 <210> SEQ IDNO 19 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer CS1 <400> SEQUENCE: 19 agcttggatc cttaagtactctagagttta aacg 34 <210> SEQ ID NO 20 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer CS2 <400>SEQUENCE: 20 aattcgttta aactctagag tacttaagga tcca 34 <210> SEQ ID NO 21<211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer MUT7 <400> SEQUENCE: 21 tcgaccctag gcacgtgacgcgtcaattgg atccgcatgc aagctt 46 <210> SEQ ID NO 22 <211> LENGTH: 46<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Primer MUT8 <400>SEQUENCE: 22 gatcaagctt gcatgcggat ccaattgacg cgtcacgtgc ctaggg 46 <210>SEQ ID NO 23 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer MUT9 <400> SEQUENCE: 23 gtgtcctcctttcacgtatt gcgatccgcc ccttcgcggt ccttcagcag cgcgcccgag 60 cgtttcagctctgccttgag gctg 84 <210> SEQ ID NO 24 <211> LENGTH: 88 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer MUT10 <400>SEQUENCE: 24 tcgacagcct caaggcagag ctgaaacgct cgggcgcgct gctgaaggaccgcgaagggg 60 cggatcgcaa tacgtgaaag gaggacac 88 <210> SEQ ID NO 25 <211>LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer MUT11 <400> SEQUENCE: 25 taagaaaccc tccttta 17 <210> SEQ ID NO 26<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer MUT12 <400> SEQUENCE: 26 ctagtaaagg agggtttct 19 <210>SEQ ID NO 27 <211> LENGTH: 11233 <212> TYPE: DNA <213> ORGANISM: Unknown<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Organism:Plasmid pZea4 <400> SEQUENCE: 27 ctaaattgta agcgttaata ttttgttaaaattcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaaaatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaacaagagtcca ctattaaaga acgtggact 180 caacgtcaaa gggcgaaaaa ccgtctatcagggcgatggc ccactacgtg aaccatcac 240 ctaatcaagt tttttggggt cgaggtgccgtaaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagccggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggcaagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctacagggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggcctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggtaacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc gcgcgtaatacgactcacta tagggcgaat tggagctcca 660 ccgcggtggc ggccgctcta gtggatccgcgcctggccgt tcgcgatcag cagccgccct 720 tgcggatcgg tcagcatcat ccccatgaaccgcagcgcac gacgcagcgc gcgccccaga 780 tcgggcgcgt ccagcacggc atgcgccatcatcgcgaagg cccccggcgg catggggcgc 840 gtgcccattc cgaagaactc gcagcctgtccgctgcgcaa ggtcgcgcca gatcgcgccg 900 tattccgatg cagtgacggg cccgatgcgcgtgggcccgc cctgccccgc cgccaccagc 960 gcatcgcgca cgaacccttc cgagatgatgtgctgatcca tggcccgtca ttgcaaaacc 1020 gatcaccgat cctgtcgcgt gatggcattgtttgcaatgc cccgagggct aggatggcgc 1080 gaaggatcaa gggggggaga gacatggaaatcgagggacg ggtctttgtc gtcacgggcg 1140 ccgcatcggg tctgggggcg gcctcggcgcggatgctggc ccaaggcggc gcgaaggtcg 1200 tgctggccga tctggcggaa ccgaaggacgcgcccgaagg cgcggttcac gcggcctgcg 1260 acgtgaccga cgcgaccgct gcgcagacggccatcgcgct ggcgaccgac cgcttcggca 1320 ggctggacgg ccttgtgaac tgcgcgggcatcgcgccggc cgaacggatg ctgggccgcg 1380 acgggccgca tggactggac agctttgcccgtgcggtcac gatcaacctg atcggcagct 1440 tcaacatggc ccgccttgca gccgaggcgatggcccggaa cgagcccgtc cggggcgagc 1500 gtggcgtgat cgtcaacacg gcctcgatcgcggcgcagga cggacagatc ggacaggtcg 1560 cctatgcggc cagcaaggcg ggcgtggcgggcatgacgct gccgatggcc cgcgaccttg 1620 cgcggcacgg catccgcgtc atgaccatcgcgcccggcat cttccgcacc ccgatgctgg 1680 aggggctgcc gcaggacgtt caggacagcctgggcgcggc ggtgcccttc ccctcgcggc 1740 tgggagagcc gtcggaatac gcggcgctgttgcaccacat catcgcgaac cccatgctga 1800 acggagaggt catccgcctc gacggcgcattgcgcatggc ccccaagtga aggagcgttt 1860 catggacccc atcgtcatca ccggcgcgatgcgcaccccg atgggggcat tccagggcga 1920 tcttgccgcg atggatgccc cgacccttggcgcggacgcg atccgcgccg cgctgaacgg 1980 cctgtcgccc gacatggtgg acgaggtgctgatgggctgc gtcctcgccg cgggccaggg 2040 tcaggcaccg gcacgtcagg cggcgcttggcgccggactg ccgctgtcga cgggcacgac 2100 caccatcaac gagatgtgcg gatcgggcatgaaggccgcg atgctgggcc atgacctgat 2160 cgccgcggga tcggcgggca tcgtcgtcgccggcgggatg gagagcatgt cgaacgcccc 2220 ctacctgctg cccaaggcgc ggtcggggatgcgcatgggc catgaccgtg tgctggatca 2280 catgttcctc gacgggttgg aggacgcctatgacaagggc cgcctgatgg gcaccttcgc 2340 cgaggattgc gccggcgatc acggtttcacccgcgaggcg caggacgact atgcgctgac 2400 cagcctggcc cgcgcgcagg acgccatcgccagcggtgcc ttcgccgccg agatcgcgcc 2460 cgtgaccgtc acggcacgca aggtgcagaccaccgtcgat accgacgaga tgcccggcaa 2520 ggcccgcccc gagaagatcc cccatctgaagcccgccttc cgtgacggtg gcacggtcac 2580 ggcggcgaac agctcgtcga tctcggacggggcggcggcg ctggtgatga tgcgccagtc 2640 gcaggccgag aagctgggcc tgacgccgatcgcgcggatc atcggtcatg cgacccatgc 2700 cgaccgtccc ggcctgttcc cgacggcccccatcggcgcg atgcgcaagc tgctggaccg 2760 cacggacacc cgccttggcg attacgacctgttcgaggtg aacgaggcat tcgccgtcgt 2820 cgccatgatc gcgatgaagg agcttggcctgccacacgat gccacgaaca tcaacggcgg 2880 ggcctgcgcg cttgggcatc ccatcggcgcgtcgggggcg cggatcatgg tcacgctgct 2940 gaacgcgatg gcggcgcggg gcgcgacgcgcggggccgca tccgtctgca tcggcggggg 3000 cgaggcgacg gccatcgcgc tggaacggctgagctaattc atttgcgcga atccgcgttt 3060 ttcgtgcacg atgggggaac cggaaacggccacgcctgtt gtggttgcgt cgacctgtct 3120 tcgggccatg cccgtgacgc gatgtggcaggcgcatgggg cgttgccgat ccggtcgcat 3180 gactgacgca acgaaggcac cgatgacgcccaagcagcaa ttccccctac gcgatctggt 3240 cgagatcagg ctggcgcaga tctcgggccagttcggcgtg gtctcggccc cgctcggcgc 3300 ggccatgagc gatgccgccc tgtcccccggcaaacgcttt cgcgccgtgc tgatgctgat 3360 ggtcgccgaa agctcgggcg gggtctgcgatgcgatggtc gatgccgcct gcgcggtcga 3420 gatggtccat gccgcatcgc tgatcttcgacgacatgccc tgcatggacg atgccaggac 3480 ccgtcgcggt cagcccgcca cccatgtcgcccatggcgag gggcgcgcgg tgcttgcggg 3540 catcgccctg atcaccgagg ccatgcggattttgggcgag gcgcgcggcg cgacgccgga 3600 tcagcgcgca aggctggtcg catccatgtcgcgcgcgatg ggaccggtgg ggctgtgcgc 3660 agggcaggat ctggacctgc acgcccccaaggacgccgcc gggatcgaac gtgaacagga 3720 cctcaagacc ggcgtgctgt tcgtcgcgggcctcgagatg ctgtccatta ttaagggtct 3780 ggacaaggcc gagaccgagc agctcatggccttcgggcgt cagcttggtc gggtcttcca 3840 gtcctatgac gacctgctgg acgtgatcggcgacaaggcc agcaccggca aggatacggc 3900 gcgcgacacc gccgcccccg gcccaaagggcggcctgatg gcggtcggac agatgggcga 3960 cgtggcgcag cattaccgcg ccagccgcgcgcaactggac gagctgatgc gcacccggct 4020 gttccgcggg gggcagatcg cggacctgctggcccgcgtg ctgccgcatg acatccgccg 4080 cagcgcctag gcgcgcggtc gggtccacaggccgtcgcgg ctgatttcgc cgccgcgcag 4140 gcgcgatgcg gccgcgtcca agcctccgcgcgccagaagc ccgatcttgg cagccttcga 4200 cgtgctgatc cgctggcgat aggcctcggggccaccctgc cggatgcgcg tcccgattgc 4260 gcgatagata cgcagcgcgg cggcgatcgaccacgcgcag cgcggcggca gatgcggaag 4320 cccctgccgc gccgaggcat aatagggctcggccgcgtca agcaggcgga tgatgacgga 4380 atagagcgcg tccgaaggca ccggaccctcaaccgtcgcc cccgcctcgg ccagccagtc 4440 ggcaggcaga tagcagcgcc cgatggcggcatcgtcgatc acgtcgcgag cgatgttcgt 4500 cagctggaac gcaaggccca gatcgcaggcgcgatccagc accgcatcgt cctgcacgcc 4560 catcacccgc gccatcatca cgcccacgacccccgcgacg tggtaggaat attccagcac 4620 gtcatccagg ctgcggtatt cgcgatccgcgacatccatc gcgaaaccct cgatcaggtc 4680 catcggccaa aggtccggga aatcatgccgccgggcgacc tggcgcagcg ccgcgaaggg 4740 cggcgacatc gggccgtcct cgtgcagcgcggccagcgtg tcggcgcgca gcgcccccag 4800 ccgcgcctgt gggtcgccgc ccgcctcgggggcagaaccc atcacctgcc cgtcgatcac 4860 gtcatccgca tgcctgcacc aggcatagagcatgaccgta tcctcgcgga tgccgggcgg 4920 catcagcttg gccgcctgcg cgaagctttgcgaaccctgc gcgatggccg cttcggaagt 4980 cgccgtcaga tcggtcatgc gacggccaggtccgacagca tgacctgcgc cgtggccttg 5040 gcgctgccaa cgacacccgg gatgcccgcacccggatgcg tgcccgcccc cacgatgtag 5100 aagttcggga tcgcgcggtc gcggttatgcgggcggaacc aggcggattg cgtcaggatc 5160 ggctcgaccg agaaggcgct gccgtgatgggccgacagtt cggtgctgaa atcggcgggg 5220 ctgaagatgc ggctgacggt caggtgcttgcgcaggtcgg ggatggcgcg gcgctccagt 5280 tcctcgaaga tgcgctcggc atagcccggggcctcggctt cccaatcgac atcggcgcgg 5340 cccagatgcg gaacgggcgc aaggacgtaatgcgtggaca tcccctcggg ggccaggctg 5400 ggatcggtca cgcagggcga atgcagatacatcgagaaat cgtccggcag gcgtggcccg 5460 ttgaagatct cgttcaccag ccccttgtagcgcgggccga agatgacgct gtggtgggcc 5520 aggttctcgg ggcgcttgga caggccgaaatgcagcacga acagcgacat cgaccagcgc 5580 tgccggttca ggatcgcggc cttggtgcgcccgcggcggg tatggcccag caggtcgcga 5640 tagctgtgca tcacgtcgcc gttgctggccaccgtatccg cgcgcaactg ccgcccgtcc 5700 agcagcgtga cgcccgtggc gcgatcgccctcggtgtcga tccgcgtgac gcgggcattc 5760 agcagcagcg tgccgccaag acgctcgaacagggcgacca tgcccgcgac cagctggttg 5820 gtgccgccct tggcgaacca gacgccgccgcgccgttcca gcgcatggat cagcgcatag 5880 atcgagctgg tcgaaaacgg gttcccgccgaccagcagcg tgtggaacga gaaggcctgc 5940 cgcagatgcg ggtcctggat gaagcgcgccaccatgctgt ggaccgagcg gtatgcctgc 6000 aggcgcatca gcgccggcgc ggcgttcagcatctggccca gcttcaggaa gggcgtggtc 6060 cccagcttca gatacccctc gcgatagacctcctcggcgt aatcgtggaa gcggcgatag 6120 ccatcgacat cggcgggatt gaaggaggcgacctggcgga tcagctcgtc gtcgtcgttc 6180 acgtattcga agctgcggcc gtccgcccatgtcagccggt agaagggcga gaccggcagc 6240 agcgtcacgt cacgctccat cggttggccgctgagggccc acagctctcg caggctgtcg 6300 gggtcggtca cgaccgtcgg gcctgcatcgaagacgtggc cctgatcgtt ccagacatag 6360 gcgcggccgc cgggcttgtc gcgggcctcgacgatggtgg tcgcgatgcc ggccgattgc 6420 aggcggatgg caagcgcaag cccgccgaaacctgcgccga tgacgatggc ggaactcatg 6480 ctctctcctg cagcaggggg cgttcgggcaggcagcgcac ggcctgcgac agcggaatgg 6540 gcgggcgtcc ggtgacgatg cgaagccggtcggccaatgt caggcgcccg gcatagaagc 6600 gctcgatcag cggctgcggc aggcggtagaaccgctgcag caggcgatag cgacggtcgg 6660 gcgggcagcc gcggaacagc atccggttcagcagccgcag gaagcggtcg cgatccgcgc 6720 gatcgatggc ccagccgcgc accgcgcgacgggcggacgc ggtcgtcagg tcgcgcgccg 6780 cgatggcatc cgcgacctgc gcggcatagggcagcgaata tccggtgacg gggtggaaca 6840 gccctgcccc cagcccaacc ggcaccgccccctgcgcgtg gtcgcgccag aagcctatgg 6900 cgtcatgggc cagcgcgatg ggcaggatgcccctttcgcg ccgcatctcc tgcccggtcc 6960 agccccgcct ggcggcatag tccagcgacgcctgcgccag cgcgccatcg tccagatcgc 7020 cgccgtcgct gtagcgcgta tcctcgatcaggatgcgggt gggactgaag ggcagcagat 7080 agatgaagcg gtacccgtcc atctgcggaacggtcgcgtc catgatcatc gggcgctcga 7140 cgccatgggg ggcgtcggtc tcgatctcgacgcccacgaa tttctggaaa cccacggtca 7200 ggtgcggggt ctcgacggca ccacgggcgtcgatcacgca ggcagcctcg atccgcgagc 7260 cgtccgtcag cgtcgcgccg gtatcgtccagcgtcgcgac atgcgtattc caccgcagat 7320 cgacaccctg cagcagcccg atcagcgcgcccgcctcgat cgagccatag cctgtcgtca 7380 ggcggcgcga atggtcggga aacgcgacctcctgatccgt ccattcgccg cgacgaatgg 7440 gcgacaggcg cgccagccat tcgggcgaaagatccgtgtc gtggcaggac caggtgtgct 7500 ggtccgaggg gccggaccgc gcgtcgagcatcacgatgcg cgcatccggt ctgcggtcgc 7560 gaacggcaag cgcgatcagc gcaccggacagccccgcgcc cgcgatcagc agatcatggc 7620 tcatgtattg cgatccgccc cttcgcggtccttcagcagc gcgcccgagc gtttcagctc 7680 tgccttgagg ctgtcgaccg agggcgcccagatgaaaccg aagctgacgc agttctcgcg 7740 gccatggacc gcgtgatgca tcctgtgtgcctggtagacg cgacgaagat agccgcgctt 7800 ggggacatag cggaacggcc agcgcccatgcaccaagccg tcatgcagga aatagtagat 7860 cagcccgtag caggtgaccc ccaccgccagccaccaggcc agatccgacc ccatcgcgcc 7920 gatcgcgaac agcacgatcg agattaccgcgaagatgacg ccatagaggt cgttcttctc 7980 gagcgcgtgg tcgtgatcct cgtcgtggtgcgatttatgc cagccccagc ccagggggcc 8040 atgcatgatc caccgatgga cggagtaggccgtcagctcc atcgcggcga cggtcaggat 8100 gacggtcagg attgcggccc aagtgctcatgccggcccct tgcttgatat gacagggaac 8160 aggctacgct gccgcgcggt gcatgaccagcccatcgggg tgcgaccaaa gggcatcgcg 8220 tgacatctgc gttcagggct cataggcggatcatccgtga cattcgccgc cgaacgcggc 8280 aggcgcatca cgcgttccgt cgctggaaatattaatgttt tcccgaagat ggtcggggcg 8340 agaggattcg aacctccgac ctacggtacccaaaaccgtc gcgctaccag gctgcgctac 8400 gccccgactg cggaaggctt tagccgattgttccggcaag ggaaagacct agtcgcaggc 8460 caggaccgca ttgtcgccca tgcccggatgcgccatcggc tgaccgggct tcaggccaag 8520 gcgatccgcc tctccgcccg cgatttcgaggacgaacagc cggtcggggt ccggatcgcc 8580 gaccgccgcg cccggaatgg gcgtctcgtccagcgggcgc gcattgcggt ggatgtggcg 8640 gatgacgccg gtttcatccg caaagaccatgtccagcggg atcagtgtgt tgcgcatcca 8700 gaaggacacc ggctggggcg attcgtagatgaacagcatt ccggtgcccg caggcagctc 8760 cttgcggaac atcaggccct gcgcgcgctcttcggggctg tccgcgacct cgacccgaaa 8820 cccgagcgtt tccgcaccgg tatcgacgacaagactgccg ggcgcgcatt ccaccgccgc 8880 cgcggcggcg ggcatcagga ccgcaagaagcgctgcggcc ttactcggcc acatgggcaa 8940 gataggactg ctcggcgccg agatcccccgggctgcagga attcgatatc aagcttatcg 9000 ataccgtcga cctcgagggg gggcccggtacccagctttt gttcccttta gtgagggtta 9060 attgcgcgct tggcgtaatc atggtcatagctgtttcctg tgtgaaattg ttatccgctc 9120 acaattccac acaacatacg agccggaagcataaagtgta aagcctgggg tgcctaatga 9180 gtgagctaac tcacattaat tgcgttgcgctcactgcccg ctttccagtc gggaaacctg 9240 tcgtgccagc tgcattaatg aatcggccaacgcgcgggga gaggcggttt gcgtattggg 9300 cgctcttccg cttcctcgct cactgactcgctgcgctcgg tcgttcggct gcggcgagcg 9360 gtatcagctc actcaaaggc ggtaatacggttatccacag aatcagggga taacgcagga 9420 aagaacatgt gagcaaaagg ccagcaaaaggccaggaacc gtaaaaaggc cgcgttgctg 9480 gcgtttttcc ataggctccg cccccctgacgagcatcaca aaaatcgacg ctcaagtcag 9540 aggtggcgaa acccgacagg actataaagataccaggcgt ttccccctgg aagctccctc 9600 gtgcgctctc ctgttccgac cctgccgcttaccggatacc tgtccgcctt tctcccttcg 9660 ggaagcgtgg cgctttctca tagctcacgctgtaggtatc tcagttcggt gtaggtcgtt 9720 cgctccaagc tgggctgtgt gcacgaaccccccgttcagc ccgaccgctg cgccttatcc 9780 ggtaactatc gtcttgagtc caacccggtaagacacgact tatcgccact ggcagcagcc 9840 actggtaaca ggattagcag agcgaggtatgtaggcggtg ctacagagtt cttgaagtgg 9900 tggcctaact acggctacac tagaaggacagtatttggta tctgcgctct gctgaagcca 9960 gttaccttcg gaaaaagagt tggtagctcttgatccggca aacaaaccac cgctggtagc 10020 ggtggttttt ttgtttgcaa gcagcagattacgcgcagaa aaaaaggatc tcaagaagat 10080 cctttgatct tttctacggg gtctgacgctcagtggaacg aaaactcacg ttaagggatt 10140 ttggtcatga gattatcaaa aaggatcttcacctagatcc ttttaaatta aaaatgaagt 10200 tttaaatcaa tctaaagtat atatgagtaaacttggtctg acagttacca atgcttaatc 10260 agtgaggcac ctatctcagc gatctgtctatttcgttcat ccatagttgc ctgactcccc 10320 gtcgtgtaga taactacgat acgggagggcttaccatctg gccccagtgc tgcaatgata 10380 ccgcgagacc cacgctcacc ggctccagatttatcagcaa taaaccagcc agccggaagg 10440 gccgagcgca gaagtggtcc tgcaactttatccgcctcca tccagtctat taattgttgc 10500 cgggaagcta gagtaagtag ttcgccagttaatagtttgc gcaacgttgt tgccattgct 10560 acaggcatcg tggtgtcacg ctcgtcgtttggtatggctt cattcagctc cggttcccaa 10620 cgatcaaggc gagttacatg atcccccatgttgtgcaaaa aagcggttag ctccttcggt 10680 cctccgatcg ttgtcagaag taagttggccgcagtgttat cactcatggt tatggcagca 10740 ctgcataatt ctcttactgt catgccatccgtaagatgct tttctgtgac tggtgagtac 10800 tcaaccaagt cattctgaga atagtgtatgcggcgaccga gttgctcttg cccggcgtca 10860 atacgggata ataccgcgcc acatagcagaactttaaaag tgctcatcat tggaaaacgt 10920 tcttcggggc gaaaactctc aaggatcttaccgctgttga gatccagttc gatgtaaccc 10980 actcgtgcac ccaactgatc ttcagcatcttttactttca ccagcgtttc tgggtgagca 11040 aaaacaggaa ggcaaaatgc cgcaaaaaagggaataaggg cgacacggaa atgttgaata 11100 ctcatactct tcctttttca atattattgaagcatttatc agggttattg tctcatgagc 11160 ggatacatat ttgaatgtat ttagaaaaataaacaaatag gggttccgcg cacatttccc 11220 cgaaaagtgc cac 11233 <210> SEQ IDNO 28 <211> LENGTH: 726 <212> TYPE: DNA <213> ORGANISM: Alcaligenes PC-1<400> SEQUENCE: 28 atgtccggtc gtaaaccggg taccaccggt gacaccatcgttaacctggg tctgaccgct 60 gctatcctgc tgtgctggct ggttctgcac gctttcaccctgtggctgct ggacgctgct 120 gctcacccgc tgctggctgt tctgtgcctg gctggtctgacctggctgtc cgttggtctg 180 ttcatcatcg ctcacgacgc tatgcacggt tccgttgttccgggtcgtcc gcgggctaac 240 gctgctatcg gtcagctggc tctgtggctg tacgctggtttctcctggcc gaaactgatc 300 gctaaacaca tgacccacca ccgtcacgct ggtaccgacaacgacccgga cttcggtcac 360 ggtggtccgg ttcgttggta cggttccttc gtttccacctacttcggttg gcgtgaaggt 420 ctgctgctgc cggttatcgt taccacctac gctctgatcctgggtgaccg ttggatgtac 480 gttatcttct ggccggttcc ggctgttctg gcttccatccagatcttcgt tttcggtacc 540 tggctgccgc accgtccggg tcacgacgac ttcccggaccgtcacaacgc tcgttccacc 600 ggtatcggtg acccgctgtc cctgctgacc tgcttccacttcggtggtta ccaccacgaa 660 caccacctgc acccgcacgt tccgtggtgg cgtctgccgcgtacccgtaa aaccggtggt 720 cgtgct 726 <210> SEQ ID NO 29 <211> LENGTH:242 <212> TYPE: PRT <213> ORGANISM: Alcaligenes PC-1 <400> SEQUENCE: 29Met Ser Gly Arg Lys Pro Gly Thr Thr Gly Asp Thr Ile Val Asn Leu 1 5 1015 Gly Leu Thr Ala Ala Ile Leu Leu Cys Trp Leu Val Leu His Ala Phe 20 2530 Thr Leu Trp Leu Leu Asp Ala Ala Ala His Pro Leu Leu Ala Val Leu 35 4045 Cys Leu Ala Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala 50 5560 His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn 65 7075 80 Ala Ala Ile Gly Gln Leu Ala Leu Trp Leu Tyr Ala Gly Phe Ser Trp 8590 95 Pro Lys Leu Ile Ala Lys His Met Thr His His Arg His Ala Gly Thr100 105 110 Asp Asn Asp Pro Asp Phe Gly His Gly Gly Pro Val Arg Trp TyrGly 115 120 125 Ser Phe Val Ser Thr Tyr Phe Gly Trp Arg Glu Gly Leu LeuLeu Pro 130 135 140 Val Ile Val Thr Thr Tyr Ala Leu Ile Leu Gly Asp ArgTrp Met Tyr 145 150 155 160 Val Ile Phe Trp Pro Val Pro Ala Val Leu AlaSer Ile Gln Ile Phe 165 170 175 Val Phe Gly Thr Trp Leu Pro His Arg ProGly His Asp Asp Phe Pro 180 185 190 Asp Arg His Asn Ala Arg Ser Thr GlyIle Gly Asp Pro Leu Ser Leu 195 200 205 Leu Thr Cys Phe His Phe Gly GlyTyr His His Glu His His Leu His 210 215 220 Pro His Val Pro Trp Trp ArgLeu Pro Arg Thr Arg Lys Thr Gly Gly 225 230 235 240 Arg Ala <210> SEQ IDNO 30 <211> LENGTH: 1261 <212> TYPE: DNA <213> ORGANISM: AlcaligenesPC-1 <400> SEQUENCE: 30 actgtagtct gcgcggatcg ccggtccggg ggacaagatatgagcgcaca tgccctgccc 60 aaggcagatc tgaccgccac cagtttgatc gtctcgggcggcatcatcgc cgcgtggctg 120 gccctgcatg tgcatgcgct gtggtttctg gacgcggcggcgcatcccat cctggcggtc 180 gcgaatttcc tggggctgac ctggctgtcg gtcggtctgttcatcatcgc gcatgacgcg 240 atgcatgggt cggtcgtgcc ggggcgcccg cgcgccaatgcggcgatggg ccagcttgtc 300 ctgtggctgt atgccggatt ttcctggcgc aagatgatcgtcaagcacat ggcccatcat 360 cgccatgccg gaaccgacga cgacccagat ttcgaccatggcggcccggt ccgctggtac 420 gcccgcttca tcggcaccta tttcggctgg cgcgaggggctgctgctgcc cgtcatcgtg 480 acggtctatg cgctgatgtt gggggatcgc tggatgtacgtggtcttctg gccgttgccg 540 tcgatcctgg cgtcgatcca gctgttcgtg ttcggcatctggctgccgca ccgccccggc 600 cacgacgcgt tcccggaccg ccacaatgcg cggtcgtcgcggatcagcga ccccgtgtcg 660 ctgctgacct gctttcactt tggcggttat catcacgaacaccacctgca cccgacggtg 720 ccttggtggc gcctgcccag cacccgcacc aagggggacaccgcatgacc aatttcctga 780 tcgtcgtcgc caccgtgctg gtgatggagc tgacggcctattccgtccac cgctggatca 840 tgcacggccc cttgggctgg ggctggcaca agtcccaccacgaggaacac gaccacgcgc 900 tggaaaagaa cgacctgtac ggcctggtct ttgcggtgatcgccacggtg ctgttcacgg 960 tgggctggat ctgggcaccg gtcctgtggt ggatcgccttgggcatgacc gtctacgggc 1020 tgatctattt cgtcctgcat gacgggctgg tgcatcagcgctggccgttc cgctatatcc 1080 ctcgcaaggg ctatgccaga cgcctgtatc aggcccaccgcctgcaccac gcggtcgagg 1140 ggcgcgacca ttgcgtcagc ttcggcttca tctatgcgccgccggtcgac aagctgaagc 1200 aggacctgaa gacgtcgggc gtgctgcggg ccgaggcgcaggagcgcacg tgacccatga 1260 c 1261 <210> SEQ ID NO 31 <211> LENGTH: 1458<212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE: <223> OTHERINFORMATION: Description of Unknown Organism: Unkown <400> SEQUENCE: 31atgagcgcac atgccctgcc caaggcagat ctgaccgcca ccagtttgat cgtctcgggc 60tactcgcgtg tacgggacgg gttccgtcta gactggcggt ggtcaaacta gcagagcccg 120ggcatcatcg ccgcgtggct ggccctgcat gtgcatgcgc tgtggtttct ggacgcggcg 180ccgtagtagc ggcgcaccga ccgggacgta cacgtacgcg acaccaaaga cctgcgccgc 240gcgcatccca tcctggcggt cgcgaatttc ctggggctga cctggctgtc ggtcggtctg 300cgcgtagggt aggaccgcca gcgcttaaag gaccccgact ggaccgacag ccagccagac 360ttcatcatcg cgcatgacgc gatgcatggg tcggtcgtgc cggggcgccc gcgcgccaat 420aagtagtagc gcgtactgcg ctacgtaccc agccagcacg gccccgcggg cgcgcggtta 480gcggcgatgg gccagcttgt cctgtggctg tatgccggat tttcctggcg caagatgatc 540cgccgctacc cggtcgaaca ggacaccgac atacggccta aaaggaccgc gttctactag 600gtcaagcaca tggcccatca tcgccatgcc ggaaccgacg acgacccaga tttcgaccat 660cagttcgtgt accgggtagt agcggtacgg ccttggctgc tgctgggtct aaagctggta 720ggcggcccgg tccgctggta cgcccgcttc atcggcacct atttcggctg gcgcgagggg 780ccgccgggcc aggcgaccat gcgggcgaag tagccgtgga taaagccgac cgcgctcccc 840ctgctgctgc ccgtcatcgt gacggtctat gcgctgatgt tgggggatcg ctggatgtac 900gacgacgacg ggcagtagca ctgccagata cgcgactaca accccctagc gacctacatg 960gtggtcttct ggccgttgcc gtcgatcctg gcgtcgatcc agctgttcgt gttcggcatc 1020caccagaaga ccggcaacgg cagctaggac cgcagctagg tcgacaagca caagccgtag 1080tggctgccgc accgccccgg ccacgacgcg ttcccggacc gccacaatgc gcggtcgtcg 1140accgacggcg tggcggggcc ggtgctgcgc aagggcctgg cggtgttacg cgccagcagc 1200cggatcagcg accccgtgtc gctgctgacc tgctttcact ttggcggtta tcatcacgaa 1260gcctagtcgc tggggcacag cgacgactgg acgaaagtga aaccgccaat agtagtgctt 1320caccacctgc acccgacggt gccttggtgg cgcctgccca gcacccgcac caagggggac 1380gtggtggacg tgggctgcca cggaaccacc gcggacgggt cgtgggcgtg gttccccctg 1440accgcatgat ggcgtact 1458 <210> SEQ ID NO 32 <211> LENGTH: 242 <212>TYPE: PRT <213> ORGANISM: E-396 <220> FEATURE: <223> OTHER INFORMATION:Description of Unknown Organism: Unkown <400> SEQUENCE: 32 Met Ser AlaHis Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu 1 5 10 15 Ile ValSer Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His 20 25 30 Ala LeuTrp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Val Ala 35 40 45 Asn PheLeu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala 50 55 60 His AspAla Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn 65 70 75 80 AlaAla Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp 85 90 95 ArgLys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr 100 105 110Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala 115 120125 Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro 130135 140 Val Ile Val Thr Val Tyr Ala Leu Met Leu Gly Asp Arg Trp Met Tyr145 150 155 160 Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile GlnLeu Phe 165 170 175 Val Phe Gly Ile Trp Leu Pro His Arg Pro Gly His AspAla Phe Pro 180 185 190 Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser AspPro Val Ser Leu 195 200 205 Leu Thr Cys Phe His Phe Gly Gly Tyr His HisGlu His His Leu His 210 215 220 Pro Thr Val Pro Trp Trp Arg Leu Pro SerThr Arg Thr Lys Gly Asp 225 230 235 240 Thr Ala <210> SEQ ID NO 33 <211>LENGTH: 972 <212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE: <223>OTHER INFORMATION: Description of Unknown Organism: Unknown <400>SEQUENCE: 33 atgaccaatt tcctgatcgt cgtcgccacc gtgctggtga tggagctgacggcctattcc 60 tactggttaa aggactagca gcagcggtgg cacgaccact acctcgactgccggataagg 120 gtccaccgct ggatcatgca cggccccttg ggctggggct ggcacaagtcccaccacgag 180 caggtggcga cctagtacgt gccggggaac ccgaccccga ccgtgttcagggtggtgctc 240 gaacacgacc acgcgctgga aaagaacgac ctgtacggcc tggtctttgcggtgatcgcc 300 cttgtgctgg tgcgcgacct tttcttgctg gacatgccgg accagaaacgccactagcgg 360 acggtgctgt tcacggtggg ctggatctgg gcaccggtcc tgtggtggatcgccttgggc 420 tgccacgaca agtgccaccc gacctagacc cgtggccagg acaccacctagcggaacccg 480 atgaccgtct acgggctgat ctatttcgtc ctgcatgacg ggctggtgcatcagcgctgg 540 tactggcaga tgcccgacta gataaagcag gacgtactgc ccgaccacgtagtcgcgacc 600 ccgttccgct atatccctcg caagggctat gccagacgcc tgtatcaggcccaccgcctg 660 ggcaaggcga tatagggagc gttcccgata cggtctgcgg acatagtccgggtggcggac 720 caccacgcgg tcgaggggcg cgaccattgc gtcagcttcg gcttcatctatgcgccgccg 780 gtggtgcgcc agctccccgc gctggtaacg cagtcgaagc cgaagtagatacgcggcggc 840 gtcgacaagc tgaagcagga cctgaagacg tcgggcgtgc tgcgggccgaggcgcaggag 900 cagctgttcg acttcgtcct ggacttctgc agcccgcacg acgcccggctccgcgtcctc 960 cgcacggcgt gc 972 <210> SEQ ID NO 34 <211> LENGTH: 162<212> TYPE: PRT <213> ORGANISM: E-396 <220> FEATURE: <223> OTHERINFORMATION: Description of Unknown Organism: Unknown <400> SEQUENCE: 34Met Thr Asn Phe Leu Ile Val Val Ala Thr Val Leu Val Met Glu Leu 1 5 1015 Thr Ala Tyr Ser Val His Arg Trp Ile Met His Gly Pro Leu Gly Trp 20 2530 Gly Trp His Lys Ser His His Glu Glu His Asp His Ala Leu Glu Lys 35 4045 Asn Asp Leu Tyr Gly Leu Val Phe Ala Val Ile Ala Thr Val Leu Phe 50 5560 Thr Val Gly Trp Ile Trp Ala Pro Val Leu Trp Trp Ile Ala Leu Gly 65 7075 80 Met Thr Val Tyr Gly Leu Ile Tyr Phe Val Leu His Asp Gly Leu Val 8590 95 His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Arg Lys Gly Tyr Ala Arg100 105 110 Arg Leu Tyr Gln Ala His Arg Leu His His Ala Val Glu Gly ArgAsp 115 120 125 His Cys Val Ser Phe Gly Phe Ile Tyr Ala Pro Pro Val AspLys Leu 130 135 140 Lys Gln Asp Leu Lys Thr Ser Gly Val Leu Arg Ala GluAla Gln Glu 145 150 155 160 Arg Thr <210> SEQ ID NO 35 <211> LENGTH:1253 <212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (911) <400> SEQUENCE: 35 ctgcaggtctgacacggcca gaaggccgcg ccgcgggccg ggggccgccg catcgcgacc 60 ggtatccttgccaagcgccg cctggtcgcc cacaacgtcc agcaggtcgt cataggactg 120 gaacacccggcccagctgac ggccaaagtc gatcatctga gtctgctcct cggcgtcgaa 180 ctccttgatcacggccagca tctccagccc ggcgatgaac agcacgccgg tcttcaggtc 240 ctgttcctgttcgacccccg cgccgttctt ggccgcgtgc aggtccaggt cctggccggc 300 gcacaggccctgcggcccca gggaccgcga caggatccgc accagctgcg cccgcaccgt 360 gcccgacgcgccgcgcgcac cggccagcag ggccatcgcc tcggtgatca gggcgatgcc 420 gcctagcacggcgcggcttt cgccatgcgc cacatgggtc gcgggctggc cgcggcgcag 480 cccggcatcgtccatgcagg gcaggtcgtc gaagatcagc gatgcggcat gcaccatctc 540 gaccgcgcaggcggcgtcga cgatcgtgtc gcagaccccg cccgaggctt ctgccgcaag 600 cagcatcagcatgccgcgga aacgcttgcc cgacgacagc gcgccatggc tcatggccgg 660 gccgagcggctgcgacacgg caccgaatcc ctgggcgatc tcctcaagtc tggtctgcag 720 aagggtggcgtggatcgggt tgacgtctcg tctcatcagt gccttcgcgc ttgggttctg 780 accaggcgggaaggtcaggc cggggcggca ccccgtgacc cgtcatccac cgtcaacagt 840 ccccatgttggaaggcttca cgcccgattg cgagcctttt cgacggcgac gcggggtcgc 900 gcggcaatttntccaacaag gtcagtggac cggcgcgccg atggccgcgc gcagccaggc 960 atccttggccggaaacaccc gcgccgcatc atgatcggcc aggatcgtcc ggcgcgcggc 1020 gcggcgcaggtcggccgcgt cacccggatt gtcaagcacc caggccatcg cgtccgcgac 1080 ctcgtccgcgtcgtccatgt cgacgatcag gccgttctcc atgtcgcgga ccagttcgcg 1140 caccggggcggtgttcgatc gatcaccagg catccggtgg ccatcgcctc ggacagggac 1200 caggaggtgacgaagggctc ggtgaaatag acatgcgcgt gcgaggcctg cag 1253 <210> SEQ ID NO 36<211> LENGTH: 1764 <212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Organism: Unknown <400>SEQUENCE: 36 atgagacgag acgtcaaccc gatccacgcc acccttctgc agaccagacttgaggagatc 60 tactctgctc tgcagttggg ctaggtgcgg tgggaagacg tctggtctgaactcctctag 120 gcccagggat tcggtgccgt gtcgcagccg ctcggcccgg ccatgagccatggcgcgctg 180 cgggtcccta agccacggca cagcgtcggc gagccgggcc ggtactcggtaccgcgcgac 240 tcgtcgggca agcgtttccg cggcatgctg atgctgcttg cggcagaagcctcgggcggg 300 agcagcccgt tcgcaaaggc gccgtacgac tacgacgaac gccgtcttcggagcccgccc 360 gtctgcgaca cgatcgtcga cgccgcctgc gcggtcgaga tggtgcatgccgcatcgctg 420 cagacgctgt gctagcagct gcggcggacg cgccagctct accacgtacggcgtagcgac 480 atcttcgacg acctgccctg catggacgat gccgggctgc gccgcggccagcccgcgacc 540 tagaagctgc tggacgggac gtacctgcta cggcccgacg cggcgccggtcgggcgctgg 600 catgtggcgc atggcgaaag ccgcgccgtg ctaggcggca tcgccctgatcaccgaggcg 660 gtacaccgcg taccgctttc ggcgcggcac gatccgccgt agcgggactagtggctccgc 720 atggccctgc tggccggtgc gcgcggcgcg tcgggcacgg tgcgggcgcagctggtgcgg 780 taccgggacg accggccacg cgcgccgcgc agcccgtgcc acgcccgcgtcgaccacgcc 840 atcctgtcgc ggtccctggg gccgcagggc ctgtgcgccg gccaggacctggacctgcac 900 taggacagcg ccagggaccc cggcgtcccg gacacgcggc cggtcctggacctggacgtg 960 gcggccaaga acggcgcggg ggtcgaacag gaacaggacc tgaagaccggcgtgctgttc 1020 cgccggttct tgccgcgccc ccagcttgtc cttgtcctgg acttctggccgcacgacaag 1080 atcgccgggc tggagatgct ggccgtgatc aaggagttcg acgccgaggagcagactcag 1140 tagcggcccg acctctacga ccggcactag ttcctcaagc tgcggctcctcgtctgagtc 1200 atgatcgact ttggccgtca gctgggccgg gtgttccagt cctatgacgacctgctggac 1260 tactagctga aaccggcagt cgacccggcc cacaaggtca ggatactgctggacgacctg 1320 gttgtgggcg accaggcggc gcttggcaag gataccggtc gcgatgcggcggcccccggc 1380 caacacccgc tggtccgccg cgaaccgttc ctatggccag cgctacgccgccgggggccg 1440 ccgcggcgcg gccttctggc cgtgtcagac ctgcagaacg tgtcccgtcactatgaggcc 1500 ggcgccgcgc cggaagaccg gcacagtctg gacgtcttgc acagggcagtgatactccgg 1560 agccgcgccc agctggacgc gatgctgcgc agcaagcgcc ttcaggctccggaaatcgcg 1620 tcggcgcggg tcgacctgcg ctacgacgcg tcgttcgcgg aagtccgaggcctttagcgc 1680 gccctgctgg aacgggttct gccctacgcc gcgcgcgcct agcgggacgaccttgcccaa 1740 gacgggatgc ggcgcgcgcg gatc 1764 <210> SEQ ID NO 37 <211>LENGTH: 293 <212> TYPE: PRT <213> ORGANISM: E-396 <220> FEATURE: <223>OTHER INFORMATION: Description of Unknown Organism: Unkown <400>SEQUENCE: 37 Met Arg Arg Asp Val Asn Pro Ile His Ala Thr Leu Leu Gln ThrArg 1 5 10 15 Leu Glu Glu Ile Ala Gln Gly Phe Gly Ala Val Ser Gln ProLeu Gly 20 25 30 Pro Ala Met Ser His Gly Ala Leu Ser Ser Gly Lys Arg PheArg Gly 35 40 45 Met Leu Met Leu Leu Ala Ala Glu Ala Ser Gly Gly Val CysAsp Thr 50 55 60 Ile Val Asp Ala Ala Cys Ala Val Glu Met Val His Ala AlaSer Leu 65 70 75 80 Ile Phe Asp Asp Leu Pro Cys Met Asp Asp Ala Gly LeuArg Arg Gly 85 90 95 Gln Pro Ala Thr His Val Ala His Gly Glu Ser Arg AlaVal Leu Gly 100 105 110 Gly Ile Ala Leu Ile Thr Glu Ala Met Ala Leu LeuAla Gly Ala Arg 115 120 125 Gly Ala Ser Gly Thr Val Arg Ala Gln Leu ValArg Ile Leu Ser Arg 130 135 140 Ser Leu Gly Pro Gln Gly Leu Cys Ala GlyGln Asp Leu Asp Leu His 145 150 155 160 Ala Ala Lys Asn Gly Ala Gly ValGlu Gln Glu Gln Asp Leu Lys Thr 165 170 175 Gly Val Leu Phe Ile Ala GlyLeu Glu Met Leu Ala Val Ile Lys Glu 180 185 190 Phe Asp Ala Glu Glu GlnThr Gln Met Ile Asp Phe Gly Arg Gln Leu 195 200 205 Gly Arg Val Phe GlnSer Tyr Asp Asp Leu Leu Asp Val Val Gly Asp 210 215 220 Gln Ala Ala LeuGly Lys Asp Thr Gly Arg Asp Ala Ala Ala Pro Gly 225 230 235 240 Pro ArgArg Gly Leu Leu Ala Val Ser Asp Leu Gln Asn Val Ser Arg 245 250 255 HisTyr Glu Ala Ser Arg Ala Gln Leu Asp Ala Met Leu Arg Ser Lys 260 265 270Arg Leu Gln Ala Pro Glu Ile Ala Ala Leu Leu Glu Arg Val Leu Pro 275 280285 Tyr Ala Ala Arg Ala 290 <210> SEQ ID NO 38 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer #7 <400>SEQUENCE: 38 cctggatgac gtgctggaat attcc 25 <210> SEQ ID NO 39 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer #8 <400> SEQUENCE: 39 caaggcccag atcgcaggcg 20 <210> SEQ ID NO 40<211> LENGTH: 391 <212> TYPE: PRT <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 40 Met Asp Pro Ile Val Ile Thr Gly Ala Met Arg ThrPro Met Gly Ala 1 5 10 15 Phe Gln Gly Asp Leu Ala Ala Met Asp Ala ProThr Leu Gly Ala Asp 20 25 30 Ala Ile Arg Ala Ala Leu Asn Gly Leu Ser ProAsp Met Val Asp Glu 35 40 45 Val Leu Met Gly Cys Val Leu Ala Ala Gly GlnGly Gln Ala Pro Ala 50 55 60 Arg Gln Ala Ala Leu Gly Ala Gly Leu Pro LeuSer Thr Gly Thr Thr 65 70 75 80 Thr Ile Asn Glu Met Cys Gly Ser Gly MetLys Ala Ala Met Leu Gly 85 90 95 His Asp Leu Ile Ala Ala Gly Ser Ala GlyIle Val Val Ala Gly Gly 100 105 110 Met Glu Ser Met Ser Asn Ala Pro TyrLeu Leu Pro Lys Ala Arg Ser 115 120 125 Gly Met Arg Met Gly His Asp ArgVal Leu Asp His Met Phe Leu Asp 130 135 140 Gly Leu Glu Asp Ala Tyr AspLys Gly Arg Leu Met Gly Thr Phe Ala 145 150 155 160 Glu Asp Cys Ala GlyAsp His Gly Phe Thr Arg Glu Ala Gln Asp Asp 165 170 175 Tyr Ala Leu ThrSer Leu Ala Arg Ala Gln Asp Ala Ile Ala Ser Gly 180 185 190 Ala Phe AlaAla Glu Ile Ala Pro Val Thr Val Thr Ala Arg Lys Val 195 200 205 Gln ThrThr Val Asp Thr Asp Glu Met Pro Gly Lys Ala Arg Pro Glu 210 215 220 LysIle Pro His Leu Lys Pro Ala Phe Arg Asp Gly Gly Thr Val Thr 225 230 235240 Ala Ala Asn Ser Ser Ser Ile Ser Asp Gly Ala Ala Ala Leu Val Met 245250 255 Met Arg Gln Ser Gln Ala Glu Lys Leu Gly Leu Thr Pro Ile Ala Arg260 265 270 Ile Ile Gly His Ala Thr His Ala Asp Arg Pro Gly Leu Phe ProThr 275 280 285 Ala Pro Ile Gly Ala Met Arg Lys Leu Leu Asp Arg Thr AspThr Arg 290 295 300 Leu Gly Asp Tyr Asp Leu Phe Glu Val Asn Glu Ala PheAla Val Val 305 310 315 320 Ala Met Ile Ala Met Lys Glu Leu Gly Leu ProHis Asp Ala Thr Asn 325 330 335 Ile Asn Gly Gly Ala Cys Ala Leu Gly HisPro Ile Gly Ala Ser Gly 340 345 350 Ala Arg Ile Met Val Thr Leu Leu AsnAla Met Ala Ala Arg Gly Ala 355 360 365 Thr Arg Gly Ala Ala Ser Val CysIle Gly Gly Gly Glu Ala Thr Ala 370 375 380 Ile Ala Leu Glu Arg Leu Ser385 390 <210> SEQ ID NO 41 <211> LENGTH: 388 <212> TYPE: PRT <213>ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 41 Asp Pro Arg LeuAla Val Arg Asp Gln Gln Pro Pro Leu Arg Ile Gly 1 5 10 15 Gln His HisPro His Glu Pro Gln Arg Thr Thr Gln Arg Ala Pro Gln 20 25 30 Ile Gly ArgVal Gln His Gly Met Arg His His Arg Glu Gly Pro Arg 35 40 45 Arg His GlyAla Arg Ala His Ser Glu Glu Leu Ala Ala Cys Pro Leu 50 55 60 Arg Lys ValAla Pro Asp Arg Ala Val Phe Arg Cys Ser Asp Gly Pro 65 70 75 80 Asp AlaArg Gly Pro Ala Leu Pro Arg Arg His Gln Arg Ile Ala His 85 90 95 Glu ProPhe Arg Asp Asp Val Leu Ile His Gly Pro Ser Leu Gln Asn 100 105 110 ArgSer Pro Ile Leu Ser Arg Asp Gly Ile Val Cys Asn Ala Pro Arg 115 120 125Ala Arg Met Ala Arg Arg Ile Lys Gly Gly Arg Asp Met Glu Ile Glu 130 135140 Gly Arg Val Phe Val Val Thr Gly Ala Ala Ser Gly Leu Gly Ala Ala 145150 155 160 Ser Ala Arg Met Leu Ala Gln Gly Gly Ala Lys Val Val Leu AlaAsp 165 170 175 Leu Ala Glu Pro Lys Asp Ala Pro Glu Gly Ala Val His AlaAla Cys 180 185 190 Asp Val Thr Asp Ala Thr Ala Ala Gln Thr Ala Ile AlaLeu Ala Thr 195 200 205 Asp Arg Phe Gly Arg Leu Asp Gly Leu Val Asn CysAla Gly Ile Ala 210 215 220 Pro Ala Glu Arg Met Leu Gly Arg Asp Gly ProHis Gly Leu Asp Ser 225 230 235 240 Phe Ala Arg Ala Val Thr Ile Asn LeuIle Gly Ser Phe Asn Met Ala 245 250 255 Arg Leu Ala Ala Glu Ala Met AlaArg Asn Glu Pro Val Arg Gly Glu 260 265 270 Arg Gly Val Ile Val Asn ThrAla Ser Ile Ala Ala Gln Asp Gly Gln 275 280 285 Ile Gly Gln Val Ala TyrAla Ala Ser Lys Ala Gly Val Ala Gly Met 290 295 300 Thr Leu Pro Met AlaArg Asp Leu Ala Arg His Gly Ile Arg Val Met 305 310 315 320 Thr Ile AlaPro Gly Ile Phe Arg Thr Pro Met Leu Glu Gly Leu Pro 325 330 335 Gln AspVal Gln Asp Ser Leu Gly Ala Ala Val Pro Phe Pro Ser Arg 340 345 350 LeuGly Glu Pro Ser Glu Tyr Ala Ala Leu Leu His His Ile Ile Ala 355 360 365Asn Pro Met Leu Asn Gly Glu Val Ile Arg Leu Asp Gly Ala Leu Arg 370 375380 Met Ala Pro Lys 385 <210> SEQ ID NO 42 <211> LENGTH: 182 <212> TYPE:PRT <213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 42 Met ThrGly Thr Arg Met Arg Arg Val Ser Arg Ile Ser Ala Pro Ser 1 5 10 15 SerPro Ile Leu Pro Met Trp Pro Ser Lys Ala Ala Ala Leu Leu Ala 20 25 30 ValLeu Met Pro Ala Ala Ala Ala Ala Val Glu Cys Ala Pro Gly Ser 35 40 45 LeuVal Val Asp Thr Gly Ala Glu Thr Leu Gly Phe Arg Val Glu Val 50 55 60 AlaAsp Ser Pro Glu Glu Arg Ala Gln Gly Leu Met Phe Arg Lys Glu 65 70 75 80Leu Pro Ala Gly Thr Gly Met Leu Phe Ile Tyr Glu Ser Pro Gln Pro 85 90 95Val Ser Phe Trp Met Arg Asn Thr Leu Ile Pro Leu Asp Met Val Phe 100 105110 Ala Asp Glu Thr Gly Val Ile Arg His Ile His Arg Asn Ala Arg Pro 115120 125 Leu Asp Glu Thr Pro Ile Pro Gly Ala Ala Val Gly Asp Pro Asp Pro130 135 140 Asp Arg Leu Phe Val Leu Glu Ile Ala Gly Gly Glu Ala Asp ArgLeu 145 150 155 160 Gly Leu Lys Pro Gly Gln Pro Met Ala His Pro Gly MetGly Asp Asn 165 170 175 Ala Val Leu Ala Cys Asp 180 <210> SEQ ID NO 43<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 43 acgaaggcac cgatgacgcc ca 22 <210> SEQ ID NO 44<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 44 cggacctggc cgtcgcatga ccatc 25 <210> SEQ ID NO45 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 45 cggatcgcaa tacatgagcc atg 23 <210> SEQ ID NO 46<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 46 ctgcaggaga gagcatgagt tccg 24 <210> SEQ ID NO47 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Flavobacterium sp.R1534 <400> SEQUENCE: 47 gcaaggggcc ggcatgagca ctt 23 <210> SEQ ID NO 48<211> LENGTH: 21 <212> TYPE: RNA <213> ORGANISM: Flavobacterium sp.<400> SEQUENCE: 48 aaaggagggu uucauaugag c 21 <210> SEQ ID NO 49 <211>LENGTH: 21 <212> TYPE: RNA <213> ORGANISM: Flavobacterium sp. <400>SEQUENCE: 49 aaaggaggac acgugaugag c 21 <210> SEQ ID NO 50 <211> LENGTH:22 <212> TYPE: RNA <213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 50aaaggaggca auugagauga gu 22 <210> SEQ ID NO 51 <211> LENGTH: 22 <212>TYPE: RNA <213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 51aaaggaggau ccaaucauga cc 22 <210> SEQ ID NO 52 <211> LENGTH: 21 <212>TYPE: RNA <213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 52aaaggagggu uucuuaugac g 21 <210> SEQ ID NO 53 <211> LENGTH: 15 <212>TYPE: RNA <213> ORGANISM: Bacillus subtilis <400> SEQUENCE: 53ucuuuccucc acuag 15 <210> SEQ ID NO 54 <211> LENGTH: 13 <212> TYPE: RNA<213> ORGANISM: Escherichia coli <400> SEQUENCE: 54 auuccuccac uag 13<210> SEQ ID NO 55 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer crtW15 <400> SEQUENCE: 55 tatatctagacatatgtccg gtcgtaaacc gg 32 <210> SEQ ID NO 56 <211> LENGTH: 40 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer crtW26 <400>SEQUENCE: 56 tatagaattc cacgtgtcaa gcacgaccac cggttttacg 40 <210> SEQ IDNO 57 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primet crtW100 <400> SEQUENCE: 57 caygaygcmatgcaygg 17 <210> SEQ ID NO 58 <211> LENGTH: 17 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Primer crtW101 <400> SEQUENCE: 58caygaygcka tgcaygg 17 <210> SEQ ID NO 59 <211> LENGTH: 17 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer crtW105 <400>SEQUENCE: 59 agrtgrtgyt crtgrtg 17 <210> SEQ ID NO 60 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Primer crtW106<400> SEQUENCE: 60 agrtgrtgyt cccartg 17 <210> SEQ ID NO 61 <211>LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primer crtW107 <400> SEQUENCE: 61 atcatatgag cgcacatgcc ctgcccaagg c 31<210> SEQ ID NO 62 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Primer crtW108 <400> SEQUENCE: 62 atctcgagtcacgtgcgctc ctgcgcctcg gcc 33 <210> SEQ ID NO 63 <211> LENGTH: 33 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer crtW113 <400>SEQUENCE: 63 atatacatat ggtgtccccc ttggtgcggg tgc 33 <210> SEQ ID NO 64<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Primer crtW114 <400> SEQUENCE: 64 tatggatccg acgcgttcccggaccgccac aatgc 35 <210> SEQ ID NO 65 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer AmpR1 <400>SEQUENCE: 65 tatatcggcc gactagtaag cttcaaaaag gatcttcacc tag 43 <210>SEQ ID NO 66 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer AmpR2 <400> SEQUENCE: 66 atatgaattcaataatattg aaaaaggaag 30

1. A process for the preparation of zeaxanthin wherein said processcomprises culturing a recombinant cell containing farnesyl pyrophosphateand isopentyl pyrophosphate under culture conditions sufficient for theexpression of enzymes which catalyze the conversion of the farnesylpyrophosphate and isopentyl pyrophosphate to zeaxanthin, saidrecombinant cell being a host cell transformed by an expression vectorcomprising a regulatory sequence and a polynucleotide containing thefollowing DNA sequences which encode said enzymes: a) a DNA sequencewhich encodes the GGPP synthase of Flavobacterium sp. R1534 (crtE) or aDNA sequence which is substantially homologous, b) a DNA sequence whichencodes the prephytoene synthase of Flavobacterium sp. R1534 (crtB) or aDNA sequence which is substantially homologous, c) a DNA sequence whichencodes the phytoene desaturase of Flavobacterium sp. R1534 (crtI) or aDNA sequence which is substantially homologous, d) a DNA sequence whichencodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY) or a DNAsequence which is substantially homologous, e) a DNA sequence whichencodes the β-carotene hydroxylase of microorganism E-396 (crtZ_(E396))or a DNA sequence which is substantially homologous; and isolating thezeaxanthin from such cells or the culture medium:
 2. The process ofclaim 1 wherein said DNA sequences are: a) a DNA sequence which encodesthe GGPP synthase of Flavobacterium sp. R1534 (crtE), b) a DNA sequencewhich encodes the prephytoene synthase of Flavobacterium sp. R1534(crtB), c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI), d) a DNA sequence which encodes thelycopene cyclase of Flavobacterium sp. R1534 (crtY), and e) a DNAsequence which encodes the β-carotene hydroxylase of microorganism E-396(crtZ_(E396)).
 3. The process of claim 2 wherein: a) the GGPP synthasehas the amino acid sequence of FIG. 8, b) the prephytoene synthase hasthe amino acid sequence of FIG. 9, c) the phytoene desaturase has theamino acid sequence of FIG. 10, d) the lycopene cyclase has the aminoacid sequence of FIG. 11, and e) the β-carotene hydroxylase has theamino acid sequence of FIG.
 34. 4. The process of claim 3 wherein: a)the DNA sequence encoding the GGPP synthase comprises bases 2521-3408 ofFIG. 7, b) the DNA sequence encoding the prephytoene synthase comprisesbases 4316-3405 of FIG. 7, c) the DNA sequence encoding the phytoenedesaturase comprises bases 4313-5797 of FIG. 7, d) the DNA sequenceencoding the lycopene cyclase comprises bases 5794-6942 of FIG. 7, ande) the DNA sequence encoding the β-carotene hydroxylase comprises thesequence of FIG.
 33. 5. A process for the preparation of canthaxanthinwherein said process comprises culturing a recombinant cell containingfarnesyl pyrophosphate and isopentyl pyrophosphate under cultureconditions sufficient for the expression of enzymes which catalyze theconversion of the farnesyl pyrophosphate and isopentyl pyrophosphate tocanthaxanthin, said recombinant cell being a host cell transformed by anexpression vector comprising a regulatory sequence and a polynucleotidecontaining the following DNA sequences which encode said enzymes: a) aDNA sequence which encodes the GGPP synthase of Flavobacterium sp. R1534(crtE) or a DNA sequence which is substantially homologous, b) a DNAsequence which encodes the prephytoene synthase of Flavobacterium sp.R1534 (crtB) or a DNA sequence which is substantially homologous, c) aDNA sequence which encodes the phytoene desaturase of Flavobacterium sp.R1534 (crtI) or a DNA sequence which is substantially homologous, d) aDNA sequence which encodes the lycopene cyclase of Flavobacterium sp.R1534 (crtY) or a DNA sequence which is substantially homologous, and e)a DNA sequence which encodes the β-carotene β4-oxygenase ofmicroorganism E-396 (crtW_(E396)) or a DNA sequence which issubstantially homologous; and isolating the canthaxanthin from suchcells or the culture medium.
 6. The process of claim 5 wherein said DNAsequences are: a) a DNA sequence which encodes the GGPP synthase ofFlavobacterium sp. R1534 (crtE), b) a DNA sequence which encodes theprephytoene synthase of Flavobacterium sp. R1534 (crtB), c) a DNAsequence which encodes the phytoene desaturase of Flavobacterium sp.R1534 (crtI), d) a DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY), and e) a DNA sequence which encodes theβ-carotene β4-oxygenase of microorganism E-396 (crtW_(E396)).
 7. Theprocess of claim 6 wherein: a) the GGPP synthase has the amino acidsequence of FIG. 8, b) the prephytoene synthase has the amino acidsequence of FIG. 9, c) the phytoene desaturase has the amino acidsequence of FIG. 10, d) the lycopene cyclase has the amino acid sequenceof FIG. 11, and e) the β-carotene β4-oxygenase has the amino acidsequence of FIG.
 32. 8. The process of claim 7 wherein: a) the DNAsequence encoding the GGPP synthase comprises bases 2521-3408 of FIG. 7,b) the DNA sequence encoding the prephytoene synthase comprises bases4316-3405 of FIG. 7, c) the DNA sequence encoding the phytoenedesaturase comprises bases 4313-5797 of FIG. 7, d) the DNA sequenceencoding the lycopene cyclase comprises bases 5794-6942 of FIG. 7, ande) the DNA sequence encoding the β-carotene β4-oxygenase comprises thesequence of FIG.
 31. 9. A process for the preparation of astaxanthin andadonixanthin wherein said process comprises culturing a recombinant cellcontaining farnesyl pyrophosphate and isopentyl pyrophosphate underculture conditions sufficient for the expression of enzymes whichcatalyze the conversion of the farnesyl pyrophosphate and isopentylpyrophosphate to astaxanthin and adonixanthin, said recombinant cellbeing a host cell transformed by an expression vector comprising aregulatory sequence and a polynucleotide containing the following DNAsequences which encode said enzymes: a) a DNA sequence which encodes theGGPP synthase of Flavobacterium sp. R1534 (crtE) or a DNA sequence whichis substantially homologous, b) a DNA sequence which encodes theprephytoene synthase of Flavobacterium sp. R1534 (crtB) or a DNAsequence which is substantially homologous, c) a DNA sequence whichencodes the phytoene desaturase of Flavobacterium sp. R1534 (crtI) or aDNA sequence which is substantially homologous, d) a DNA sequence whichencodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY) or a DNAsequence which is substantially homologous, e) a DNA sequence whichencodes the β-carotene β4-oxygenase of Alcaligenes PC-1 (crtW) or a DNAsequence which is substantially homologous, and f) a DNA sequence whichencodes the β-carotene hydroxylase of microorganism E-396 (crtZ_(E396))or a DNA sequence which is substantially homologous; and isolating theastaxanthin and adonixanthin from such cells or the culture medium. 10.The process of claim 9 wherein said DNA sequences are: a) a DNA sequencewhich encodes the GGPP synthase of Flavobacterium sp. R1534 (crtE), b) aDNA sequence which encodes the prephytoene synthase of Flavobacteriumsp. R1534 (crtB), c) a DNA sequence which encodes the phytoenedesaturase of Flavobacterium sp. R1534 (crtI), d) a DNA sequence whichencodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY), e) aDNA sequence which encodes the β-carotene β4-oxygenase of AlcaligenesPC-1 (crtW), and f) a DNA sequence which encodes the β-carotenehydroxylase of microorganism E-396 (crtZ_(E396)).
 11. The process ofclaim 10 wherein: a) the GGPP synthase has the amino acid sequence ofFIG. 8, b) the prephytoene synthase has the amino acid sequence of FIG.9, c) the phytoene desaturase has the amino acid sequence of FIG. 10, d)the lycopene cyclase has the amino acid sequence of FIG. 11, e) theβ-carotene β4-oxygenase has the amino acid sequence of FIG. 25, and f)the β-carotene hydroxylase has the amino acid sequence of FIG.
 34. 12.The process of claim 11 wherein: a) the DNA sequence encoding the GGPPsynthase comprises bases 2521-3408 of FIG. 7, b) the DNA sequenceencoding the prephytoene synthase comprises bases 4316-3405 of FIG. 7,c) the DNA sequence encoding the phytoene desaturase comprises bases4313-5797 of FIG. 7, d) the DNA sequence encoding the lycopene cyclasecomprises bases 5794-6942 of FIG. 7, e) the DNA sequence encoding theβ-carotene β4-oxygenase comprises the sequence of FIG. 25, and f) theDNA sequence encoding the β-carotene hydroxylase comprises the sequenceof FIG.
 33. 13. A process for the preparation of astaxanthin andadonixanthin wherein said process comprises culturing a recombinant cellcontaining farnesyl pyrophosphate and isopentyl pyrophosphate underculture conditions sufficient for the expression enzymes which catalyzethe conversion of the farnesyl pyrophosphate and isopentyl pyrophosphateto astaxanthin and adonixanthin, said recombinant cell being a host celltransformed by an expression vector comprising a regulatory sequence anda polynucleotide containing the following DNA sequences which encodesaid enzymes: a) a DNA sequence which encodes the GGPP synthase ofFlavobacterium sp. R1534 (crtE) or a DNA sequence which is substantiallyhomologous, b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) or a DNA sequence which is substantiallyhomologous, c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) or a DNA sequence which is substantiallyhomologous, d) a DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) or a DNA sequence which is substantiallyhomologous, e) a DNA sequence which encodes the b-carotene b4-oxygenaseof microorganism E-396 (crtW_(E396)) or a DNA sequence which issubstantially homologous, and f) a DNA sequence which encodes theβ-carotene hydroxylase of microorganism E-396 (crtZ_(E396)) or a DNAsequence which is substantially homologous; and isolating theastaxanthin and adonixanthin from such cells or the culture medium. 14.The process of claim 13 wherein said DNA sequences are: a) a DNAsequence which encodes the GGPP synthase of Flavobacterium sp. R1534(crtE), b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB), c) a DNA sequence which encodes thephytoene desaturase of Flavobacterium sp. R1534 (crtI), d) a DNAsequence which encodes the lycopene cyclase of Flavobacterium sp. R1534(crtY), e) a DNA sequence which encodes the β-carotene β4-oxygenase ofmicroorganism E-396 (crtW_(E396)), and f) a DNA sequence which encodesthe β-carotene hydroxylase of microorganism E-396 (crtZ_(E396)).
 15. Theprocess of claim 14 wherein: a) the GGPP synthase has the amino acidsequence of FIG. 8, b) the prephytoene synthase has the amino acidsequence of FIG. 9, c) the phytoene desaturase has the amino acidsequence of FIG. 10, d) the lycopene cyclase has the amino acid sequenceof FIG. 11, e) the β-carotene β4-oxygenase has the amino acid sequenceof FIG. 32, and f) the β-carotene hydroxylase has the amino acidsequence of FIG.
 34. 16. The process of claim 15 wherein: a) the DNAsequence encoding the GGPP synthase comprises bases 2521-3408 of FIG. 7,b) the DNA sequence encoding the prephytoene synthase comprises bases4316-3405 of FIG. 7, c) the DNA sequence encoding the phytoenedesaturase comprises bases 4313-5797 of FIG. 7, d) the DNA sequenceencoding the lycopene cyclase comprises bases 5794-6942 of FIG. 7, e)the DNA sequence encoding the β-carotene β4-oxygenase comprises thesequence of FIG. 31, and f) the DNA sequence encoding the β-carotenehydroxylase comprises the sequence of FIG.
 33. 17. A process for thepreparation of adonixanthin wherein said process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to adonixanthin, said recombinant cell being ahost cell transformed by an expression vector comprising a regulatorysequence and a polynucleotide containing the following DNA sequenceswhich encode said enzymes: a) a DNA sequence which encodes the GGPPsynthase of microorganism E-396 (crtE_(E396)) or a DNA sequence which issubstantially homologous, b) a DNA sequence which encodes theprephytoene synthase of microorganism E-396 (crtB_(E396)) or a DNAsequence which is substantially homologous, c) a DNA sequence whichencodes the phytoene desaturase of microorganism E-396 (crtI_(E396)) ora DNA sequence which is substantially homologous, d) a DNA sequencewhich encodes the lycopene cyclase of microorganism E-396 (crtY_(E396))or a DNA sequence which is substantially homologous, e) a DNA sequencewhich encodes the b-carotene b4-oxygenase of microorganism E-396(crtW_(E396)) or a DNA sequence which is substantially homologous, andf) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) or a DNA sequence which issubstantially homologous, said host cell being substantially free ofother polynucleotides of microorganism E-396; and isolating theadonixanthin from such cells or the culture medium.
 18. The process ofclaim 17 wherein said DNA sequences are: a) a DNA sequence which encodesthe GGPP synthase of microorganism E-396 (crtE_(E396)), b) a DNAsequence which encodes the prephytoene synthase of microorganism E-396(crtB_(E396)), c) a DNA sequence which encodes the phytoene desaturaseof microorganism E-396 (crtI_(E396)), d) a DNA sequence which encodesthe lycopene cyclase of microorganism E-396 (crtY_(E396)), e) a DNAsequence which encodes the β-carotene β4-oxygenase of microorganismE-396 (crtW_(E396)), and f) a DNA sequence which encodes the β-carotenehydroxylase of microorganism E-396 (crtZ_(E396)).
 19. The process ofclaim 18 wherein the polynucleotide is plasmid pE396CARcrtW-E.